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https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3861
Buffer optimization
2011-10-05T17:21:39Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into 13 commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', 46, 11-22.</ref><br><br />
<br />
<br />
<br />
==== '''NMR screening and optimization buffers:''' ====<br />
<br />
MJ001&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl, 5mM CaCl<sub>2</sub>, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1x Proteinase Inhibitors, 5% D<sub>2</sub>O <br />
<br />
MJ002&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM NH<sub>4</sub>OAc, pH 5.5, 100mM NaCl, 5mM CaCl<sub>2</sub>, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br> <br />
<br />
MJ003&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM NH<sub>4</sub>OAc, pH 4.5, 100mM NaCl, 5mM CaCl<sub>2</sub>, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br> <br />
<br />
MJ004&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM NH<sub>4</sub>OAc, pH 5.0, 10mM DTT, 50mM Arginine, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O <br />
<br />
MJ005&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM NH<sub>4</sub>OAc, pH 5.0, 10mM DTT, 5% Acetonitrile, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O <br />
<br />
MJ006&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM MES, pH 6.0, 10mM DTT, 50mM Arginine, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O <br />
<br />
MJ007&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM MES, pH 6.0, 10mM DTT, 5% Acetonitrile, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O <br />
<br />
MJ008&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 25mM Na<sub>2</sub>PO<sub>4</sub>, pH 6.5, 450mM NaCl, 10mM DTT, 20uM ZnSO<sub>4</sub>, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O <br />
<br />
MJ009&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl, 5% Acetonitrile, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O <br />
<br />
MJ010&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl, 50mM Arginine, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O <br />
<br />
MJ011&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp; 20mM MES, pH 6.5, 100mM NaCl, 1% Zwitter, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O <br />
<br />
MJ012&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl,&nbsp; 50uM ZnSO4, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O <br />
<br />
Low Salt&nbsp;&nbsp;&nbsp;&nbsp; 10mM Tris-HCl, pH 7.5, 5mM DTT, 100mM NaCl , 0.02% NaN<sub>3</sub>, 50 uM DSS, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br> <br />
<br />
<br> <br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitors: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
DSS: 2,2-Dimethyl-2-silapentane-5-sulfonic acid <br />
<br />
==== <br> <br> References ====<br />
<br />
<references /></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3860
Buffer optimization
2011-10-05T17:20:57Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into 13 commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', 46, 11-22.</ref><br> <br />
====<br />
<br />
==== '''NMR screening and optimization buffers:''' ====<br />
<br />
MJ001&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl, 5mM CaCl<sub>2</sub>, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1x Proteinase Inhibitors, 5% D<sub>2</sub>O<br />
<br />
MJ002&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM NH<sub>4</sub>OAc, pH 5.5, 100mM NaCl, 5mM CaCl<sub>2</sub>, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br><br />
<br />
MJ003&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM NH<sub>4</sub>OAc, pH 4.5, 100mM NaCl, 5mM CaCl<sub>2</sub>, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br><br />
<br />
MJ004&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM NH<sub>4</sub>OAc, pH 5.0, 10mM DTT, 50mM Arginine, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O<br />
<br />
MJ005&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM NH<sub>4</sub>OAc, pH 5.0, 10mM DTT, 5% Acetonitrile, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O<br />
<br />
MJ006&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM MES, pH 6.0, 10mM DTT, 50mM Arginine, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O<br />
<br />
MJ007&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM MES, pH 6.0, 10mM DTT, 5% Acetonitrile, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O<br />
<br />
MJ008&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 25mM Na<sub>2</sub>PO<sub>4</sub>, pH 6.5, 450mM NaCl, 10mM DTT, 20uM ZnSO<sub>4</sub>, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O<br />
<br />
MJ009&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl, 5% Acetonitrile, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br />
<br />
MJ010&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl, 50mM Arginine, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br />
<br />
MJ011&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp; 20mM MES, pH 6.5, 100mM NaCl, 1% Zwitter, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br />
<br />
MJ012&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl,&nbsp; 50uM ZnSO4, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br />
<br />
Low Salt&nbsp;&nbsp;&nbsp;&nbsp; 10mM Tris-HCl, pH 7.5, 5mM DTT, 100mM NaCl , 0.02% NaN<sub>3</sub>, 50 uM DSS, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br><br />
<br />
<br> <br />
<br><br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitors: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
DSS: 2,2-Dimethyl-2-silapentane-5-sulfonic acid <br />
<br />
==== <br> <br> References ====<br />
<br />
<references /></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=NMR_determined_Rotational_correlation_time&diff=3859
NMR determined Rotational correlation time
2011-10-05T17:04:24Z
<p>Jma: </p>
<hr />
<div>[[Image:T1wiki fig5.png|right|400px]] Brownian rotation diffusion of a particle in solution has a characteristic time constant called rotational correlation time (τ<sub>c</sub>). It is the time it takes the particle to rotate by one radian and it depends on the particle size. For globular proteins a spherical approximation can be used and the rotational correlation time is given by Stoke's law <br />
<br />
:<math>\tau_c=\frac{4\pi\eta r^3}{3kT}</math>,<br />
<br />
where <span class="texhtml">η</span> is the viscosity of the solvent, <span class="texhtml">''r''</span> is the effective hydrodynamic radius of the protein molecule, <span class="texhtml">''k''</span> is the Boltzmann constant and <span class="texhtml">''T''</span> is the temperature. The hydrodynamic radius can be estimated from the molecular weight of the protein (M) as <br />
<br />
:<math>r\approx\sqrt[3]{\frac{3M}{4\pi\rho N_a}}+r_w</math>,<br />
<br />
where <span class="texhtml">ρ</span> is the average density for proteins (1.37 g/cm3), <span class="texhtml">''N''<sub>''a''</sub></span> is the Avogadro's number and <span class="texhtml">''r''<sub>''w''</sub></span> is the hydration radius (1.6 to 3.2 A)<ref>Cavanagh J., Fairbrother W.J., Palmer, A.G, Rance M., Skelton N.J. (2007) Protein NMR Spectroscopy: Principles and Practice, p21, Elsevier</ref>. As a general rule of thumb, the τ<sub>c</sub> of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kDa. <br />
<br />
<br> <br />
<br />
For rigid protein molecules, in the limit of slow molecular motion (τ<sub>c</sub> &gt;&gt; 0.5 ns) and high magnetic field (500 MHz or greater), a closed-form solution for τ<sub>c</sub> as a function of the ratio of the longitudinal (T<sub>1</sub>) and transverse (T<sub>2</sub>) <sup>15</sup>N relaxation times exists: <br />
<br />
:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,<br />
<br />
where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in ref.<ref>Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by &amp;lt;sup&amp;gt;15&amp;lt;/sup&amp;gt;N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. ''Biochemistry'', '''28''', 8972-8979.</ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms. <br />
<br />
<br> Average <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for a given protein can be measured using 1D <sup>15</sup>N-edited relaxation experiments <ref>Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay, L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by &amp;lt;sup&amp;gt;15&amp;lt;/sup&amp;gt;N NMR relaxation. ''Biochemistry'', '''33''', 5984-6003.</ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide <sup>1</sup>H region (typically 10.5 to 8.5 ppm) and the results are used to fit an exponential decay as a function of delay time. One then computes the correlation time using Eq. 3, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T<sub>1</sub>/T<sub>2</sub> method is suitable for proteins with molecular weight of up to MW ≈ 25 kDa. Accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes difficult for larger proteins and cross-correlated relaxation rates are measured instead. <br />
<br />
<br> <br />
<br />
Below is a table of rotational correlation time values compiled for known monomeric NESG&nbsp;targets (J. Aramini; June 2010).&nbsp; All data was recorded on a Bruker 600 NMR instrument at 298 K.&nbsp; The molecular weight for each target takes into account isotopic enrichment and the presence of affinity purification tags (if any). <br />
<br />
<br> <br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" style="width: 1047px; height: 393px;"<br />
|-<br />
| width="250" align="center" | '''NESG target (isotope labeling)''' <br />
| align="center" | '''MW (kDa)''' <br />
| align="center" | '''<sup>15</sup>N ''T<sub></sub>''<sub>1</sub> (ms)''' <br />
| align="center" | '''<sup>15</sup>N ''T''<sub>2</sub> (ms)''' <br />
| align="center" | '''τ<sub>c</sub> (ns)'''<br />
|-<br />
| width="250" align="center" | PsR76A&nbsp; (NC5) <br />
| align="center" | 7.2 <br />
| align="center" | 478.0 <br />
| align="center" | 128.0 <br />
| align="center" | 5.10<br />
|-<br />
| width="250" align="center" | VfR117 (NC) <br />
| align="center" | 11.2 <br />
| align="center" | 605.0 <br />
| align="center" | 119.0 <br />
| align="center" | 6.30<br />
|-<br />
| width="250" align="center" | SyR11 (NC5) <br />
| align="center" | 12.4 <br />
| align="center" | 630.0 <br />
| align="center" | 104.0 <br />
| align="center" | 7.10<br />
|-<br />
| width="250" align="center" | ER541-37-162 (NC5) <br />
| align="center" | 15.8 <br />
| align="center" | 729.0 <br />
| align="center" | 66.5 <br />
| align="center" | 10.0<br />
|-<br />
| width="250" align="center" | ER540 (NC5) <br />
| align="center" | 18.8 <br />
| align="center" | 909.0 <br />
| align="center" | 66.5 <br />
| align="center" | 11.3<br />
|-<br />
| width="250" align="center" | SoR190 (NC) <br />
| align="center" | 13.8 <br />
| align="center" | 697.5 <br />
| align="center" | 100.9 <br />
| align="center" | 7.70<br />
|-<br />
| width="250" align="center" | TR80 (NC5) <br />
| align="center" | 10.5 <br />
| align="center" | 612.8 <br />
| align="center" | 102.9 <br />
| align="center" | 7.00<br />
|-<br />
| width="250" align="center" | Ubiquitin (NC) <br />
| align="center" | 9.0 <br />
| align="center" | 441.8 <br />
| align="center" | 144.6 <br />
| align="center" | 4.40<br />
|-<br />
| width="250" align="center" | HR2873B&nbsp;(NC) <br />
| align="center" | 10.7 <br />
| align="center" | 492.0 <br />
| align="center" | 115.0 <br />
| align="center" | 5.70<br />
|-<br />
| width="250" align="center" | B-domain (NC) <br />
| align="center" | 7.2 <br />
| align="center" | 423.5 <br />
| align="center" | 153.3 <br />
| align="center" | 4.05<br />
|-<br />
| width="250" align="center" | BcR97A&nbsp;(NC) <br />
| align="center" | 13.1 <br />
| align="center" | 705.8 <br />
| align="center" | 80.6 <br />
| align="center" | 8.80<br />
|-<br />
| width="250" align="center" | PfR193A (NC) <br />
| align="center" | 13.6 <br />
| align="center" | 733.9 <br />
| align="center" | 80.9 <br />
| align="center" | 9.00<br />
|-<br />
| width="250" align="center" | MvR76 (NC) <br />
| align="center" | 20.2 <br />
| align="center" | 1015.0 <br />
| align="center" | 64.5 <br />
| align="center" | 12.2<br />
|-<br />
| width="250" align="center" | DvR115G (NC) <br />
| align="center" | 10.9 <br />
| align="center" | 608.7 <br />
| align="center" | 115.6 <br />
| align="center" | 6.50<br />
|-<br />
| width="250" align="center" | MrR110B (NC5) <br />
| align="center" | 11.8 <br />
| align="center" | 707.0 <br />
| align="center" | 99.2 <br />
| align="center" | 7.80<br />
|-<br />
| width="250" align="center" | VpR247 (NC5) <br />
| align="center" | 12.5 <br />
| align="center" | 661.2 <br />
| align="center" | 88.3 <br />
| align="center" | 8.05<br />
|-<br />
| width="250" align="center" | BcR147A (NC) <br />
| align="center" | 11.9 <br />
| align="center" | 645.0 <br />
| align="center" | 104.0 <br />
| align="center" | 7.20<br />
|-<br />
| width="250" align="center" | WR73 (NC5) <br />
| align="center" | 21.9 <br />
| align="center" | 1261.0 * <br />
| align="center" | 41.3 * <br />
| align="center" | 13.0<br />
|-<br />
| width="250" align="center" | NsR431C (NC5) <br />
| align="center" | 16.8 <br />
| align="center" | 855.5 <br />
| align="center" | 71.2 <br />
| align="center" | 10.6<br />
|-<br />
| width="250" align="center" | StR82 (NC) <br />
| align="center" | 9.2 <br />
| align="center" | 537.3 <br />
| align="center" | 100.4 <br />
| align="center" | 6.6<br />
|}<br />
<br />
*data obtained on an 800 MHz Bruker spectrometer at 298 K.<br />
<br />
<br> <br />
<br />
== <span class="mw-headline">'''Protocols for Bruker and Varian NMR Instruments'''</span> ==<br />
<br />
*[[Measuring 15N T1 and T2 Relaxation Times on Bruker Instruments|Bruker protocol]] <br />
*[[Estimation of rotational correlation time|Varian protocol]]<br />
<br />
<br> <br />
<br />
== <span class="mw-headline">'''References''' </span> ==<br />
<br />
<references /></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3858
Buffer optimization
2011-10-05T16:23:04Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', 46, 11-22.</ref><br> <br />
<br />
==== '''NMR screening buffers:''' ====<br />
<br />
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
<br> <br />
<br />
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O. <br />
<br />
<br> <br />
<br />
TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. <br />
<br />
<br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
TCEP: ''tris''(2-carboxyethyl)phosphine <br />
<br />
==== <br> <br> References ====<br />
<br />
<references /></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3857
Buffer optimization
2011-10-05T16:22:32Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', 46, 11-22.</ref><br> <br />
<br />
==== '''NMR screening buffers:''' ====<br />
<br />
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
<br> <br />
<br />
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O. <br />
<br />
<br> <br />
<br />
TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. <br />
<br />
<br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
TCEP: ''tris''(2-carboxyethyl)phosphine <br />
<br />
==== <br> <br> References ====<br />
<br />
[<references />]</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3856
Buffer optimization
2011-10-05T16:21:01Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', 46, 11-22.</ref><br> <br />
<br />
==== '''NMR screening buffers:''' ====<br />
<br />
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
<br> <br />
<br />
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O. <br />
<br />
<br> <br />
<br />
TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. <br />
<br />
<br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
TCEP: ''tris''(2-carboxyethyl)phosphine <br />
<br />
==== <br> <br> References ====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19915800 <references />]</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3855
Buffer optimization
2011-10-05T16:20:14Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', 46, 11-22.</ref><br> <br />
<br />
==== '''NMR screening buffers:''' ====<br />
<br />
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
<br> <br />
<br />
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O. <br />
<br />
<br> <br />
<br />
TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. <br />
<br />
<br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
TCEP: ''tris''(2-carboxyethyl)phosphine <br />
<br />
==== <br> <br> References ====<br />
<br />
<references /></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3854
Buffer optimization
2011-10-05T16:19:26Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''46, 11-22.</ref><br> <br />
<br />
==== '''NMR screening buffers:''' ====<br />
<br />
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
<br> <br />
<br />
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O. <br />
<br />
<br> <br />
<br />
TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. <br />
<br />
<br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
TCEP: ''tris''(2-carboxyethyl)phosphine <br />
<br />
==== <br> <br> References ====<br />
<br />
<references /></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3853
Buffer optimization
2011-10-05T16:18:11Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''in press.</ref><br> <br />
<br />
==== '''NMR screening buffers:''' ====<br />
<br />
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
<br> <br />
<br />
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O. <br />
<br />
<br> <br />
<br />
TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. <br />
<br />
<br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
TCEP: ''tris''(2-carboxyethyl)phosphine <br />
<br />
==== <br> <br> References ====<br />
<br />
<references /></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3852
Buffer optimization
2011-10-05T16:12:45Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''in press.</ref><br> <br />
<br />
==== '''NMR screening buffers:''' ====<br />
<br />
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
<br> <br />
<br />
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O. <br />
<br />
<br> <br />
<br />
TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. <br />
<br />
<br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
TCEP: ''tris''(2-carboxyethyl)phosphine <br />
<br />
==== <br> <br> References ====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19915800 ]<references /></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3851
Buffer optimization
2011-10-05T16:11:36Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''in press.</ref><br> <br />
<br />
==== '''NMR screening buffers:''' ====<br />
<br />
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
<br> <br />
<br />
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O. <br />
<br />
<br> <br />
<br />
TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. <br />
<br />
<br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
TCEP: ''tris''(2-carboxyethyl)phosphine <br />
<br />
==== <br> <br> References ====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19915800 1. Rossi, P. Swapna, G.V.T., Huang, Y.J., Aramini, J.M., Anklin, C., Conover, K., Hamilton, K., Xiao, R., Acton, T.B., Ertekin, A., Everett, J.K., and Montelione, G.T.&nbsp; A microscale protein NMR sample screening pipeline.&nbsp; (2010)&nbsp; ''J. Biomol. NMR 46'', 11-22.]&nbsp;</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3850
Buffer optimization
2011-10-05T16:10:15Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''in press.</ref><br> <br />
<br />
==== '''NMR screening buffers:''' ====<br />
<br />
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
<br> <br />
<br />
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O. <br />
<br />
<br> <br />
<br />
TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. <br />
<br />
<br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
TCEP: ''tris''(2-carboxyethyl)phosphine <br />
<br />
==== <br> <br> References ====<br />
<br />
1. Rossi, P. Swapna, G.V.T., Huang, Y.J., Aramini, J.M., Anklin, C., Conover, K., Hamilton, K., Xiao, R., Acton, T.B., Ertekin, A., Everett, J.K., and Montelione, G.T.&nbsp; A microscale protein NMR sample screening pipeline.&nbsp; (2010)&nbsp; ''J. Biomol. NMR 46'', 11-22.&nbsp;</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Buffer_optimization&diff=3849
Buffer optimization
2011-10-05T16:09:10Z
<p>Jma: </p>
<hr />
<div>Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:<br> <br />
<br />
#slow precipitation <br />
#mixture of folded and unfolded protein <br />
#aggregation problems <br />
#multiple populations (too many peaks) for other reasons <br />
#and many other reasons<br />
<br />
<br> <br />
<br />
At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''in press.</ref><br> <br />
<br />
==== '''NMR screening buffers:''' ====<br />
<br />
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter <br />
<br />
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x <br />
<br />
<br> <br />
<br />
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O. <br />
<br />
<br> <br />
<br />
TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. <br />
<br />
<br> <br />
<br />
==== '''Abbreviations:''' ====<br />
<br />
DTT: Dithiothreitol <br />
<br />
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM) <br />
<br />
MES: 2-(''N''-morpholino)ethanesulfonic acid <br />
<br />
Tris: tris(hydroxymethyl)aminomethane; <br />
<br />
Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE); <br />
<br />
TCEP: ''tris''(2-carboxyethyl)phosphine <br />
<br />
==== <br> <br> References ====<br />
<br />
[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Rossi, P. Swapna, G.V.T., Huang, Y.J., Aramini, J.M., Anklin, C., Conover, K., Hamilton, K., Xiao, R., Acton, T.B., Ertekin, A., Everett, J.K., and Montelione, G.T.&nbsp; A microscale protein NMR sample screening pipeline.&nbsp; (2010)&nbsp; ''J. Biomol. NMR 46'', 11-22.&nbsp;][http://www3.interscience.wiley.com/journal/114029977/abstract ][http://www3.interscience.wiley.com/journal/114029977/abstract ]</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=AGNuS/AutoProc&diff=3848
AGNuS/AutoProc
2011-10-05T15:57:30Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
[http://www-nmr.cabm.rutgers.edu/software/autoproc.htm Autoproc/AGNuS] is a suite of programs for the automatic generation of scripts for multidimensional protein NMR data processing and format conversion. AGNuS automatically generates NMRPipe scripts for processing multidimensional Varian or Bruker NMR experiments and provides functions for chemical referencing.&nbsp; AGNuS calls information&nbsp; for specific experiments and spectrometers stored in table files, which can be generated/modified by users with permission.&nbsp; AGNuS is also capable of processing reduced dimensionality/GFT experiments.&nbsp; <br />
<br />
The current version of the program is AutoProc 2.0.&nbsp; Please check the [[Media:AGNuS_help.jpg|documentation]] and examples provided with the software for addtional information.<br> <br />
<br />
== '''How to Use AGNuS''' ==<br />
<br />
=== Processing a 2D <sup>1</sup>H,<sup>15</sup>N-HSQC Using AGNuS ===<br />
<br />
AGNuS parses procpar (Varian) and acqu* (Bruker) files for spectral parameters.&nbsp; The program consults a pulse sequence table specific to the sequence used in the experiment.&nbsp; For example, this is the table file for the standard Bruker 2D <sup>1</sup>H,<sup>15</sup>N-HSQC, hsqcetf3gpsi.&nbsp; If the sequence table file is missing from the Pulse_Sequences directory in AGNuS, the program will throw an error and the user must create one (best to clone one from an similar one already in the directory).<br> <br />
<pre># This is a Bruker experiment and only a table for testing.<br />
<br />
file_type&nbsp;: single_fid_table<br />
name&nbsp;: hsqcetf3gpsi<br />
nickname&nbsp;: HSQC<br />
dimensions&nbsp;: [<br />
nuclei&nbsp;: H<br />
offset_parameter&nbsp;: O1_1<br />
acquisition_mode&nbsp;: DQD<br />
]<br />
dimensions&nbsp;: [<br />
nuclei&nbsp;: N<br />
offset_parameter&nbsp;: O1_2<br />
acquisition_mode&nbsp;: Echo-AntiEcho<br />
]<br />
<br />
processing_defaults&nbsp;: [<br />
&nbsp; name&nbsp;: "Sine Bell Default"<br />
&nbsp; suffix&nbsp;: _SP<br />
&nbsp; dimensions&nbsp;:&nbsp; [<br />
&nbsp;&nbsp;&nbsp; transformation&nbsp;: FT<br />
&nbsp;&nbsp;&nbsp; apodization&nbsp;: "SP -off 0.45 -end 0.98 -pow 2"<br />
&nbsp;&nbsp;&nbsp; first_point_correction&nbsp;: 0.5<br />
&nbsp;&nbsp;&nbsp; phase0&nbsp;: -60<br />
&nbsp;&nbsp;&nbsp; ]<br />
&nbsp; dimensions&nbsp;:&nbsp; [<br />
&nbsp;&nbsp;&nbsp; transformation&nbsp;: FT<br />
&nbsp;&nbsp;&nbsp; apodization&nbsp;: "SP -off 0.45 -end 0.98 -pow 2"<br />
&nbsp;&nbsp;&nbsp; first_point_correction&nbsp;: 0.5<br />
&nbsp;&nbsp;&nbsp; phase0&nbsp;: -90<br />
&nbsp;&nbsp;&nbsp; ]<br />
&nbsp; ]<br />
<br />
</pre> <br />
Start autoproc and enter a spectrometer, then a DSS value for referencing.&nbsp; This is the most optimal way to reference a spectrum.&nbsp; We reference to internal DSS in all of our production work.&nbsp; In a typical data production run collection we start with a 1D <sup>1</sup>H NMR spectrum and obtain the absolute resonance frequency of DSS under the conditions used for the NMR experiments.&nbsp; This value is then used in the subsequent AutoProc processing of all fids from the data collection, as described below (option 7). <br />
<pre>AGNuS 2.1<br />
&nbsp; Type "agnus -help" for help<br />
&nbsp; Documentation is located at URL file:/farm/software/NewAutoProc2.0/help_files/index.html<br />
<br />
<br />
WARNING *** SW_h (2000) does not equal SW * SFO1 (4865.4880848234). Setting SW_h = SW * SFO1.<br />
<br />
Experiment hsqcetf3gpsi selected.<br />
<br />
<br />
** Spectrometer Selection Menu **<br />
<br />
1) CABM_Bruker_500<br />
2) CABM_Bruker_600_100306<br />
3) CABM_Varian_500<br />
4) CABM_Varian_500_060805_20C<br />
5) CABM_Varian_600<br />
6) NYSBC_800_Conventional<br />
7) PNNL_Baker_600<br />
8) PNNL_Glacier_600<br />
9) PNNL_Varian_500<br />
10) PNNL_Varian_750<br />
11) PNNL_Varian_800<br />
12) SUNY_Buffalo_Varian_600<br />
13) SUNY_Buffalo_Varian_750<br />
14) Z_500_No_Reference<br />
15) Z_600_No_Reference<br />
16) Z_750_No_Reference<br />
17) Z_800_No_Reference<br />
<br />
Spectrometer [1]: 17<br />
<br />
** Conversion Parameter Selection Menu **<br />
<br />
-- Current Directory: /farm/data/jma/JIM_NESG_PROJECTS/PfR193A_PROJECT/FIDs/B800/PfR193A.019_NC5_062509/NHhsqc_largeSW1_exp4<br />
-- Input Directory: ./<br />
1) Input Data File (FID) Name: ser<br />
2) Working Directory: ./<br />
-- Conversion Subdirectory: pipe_conversion/<br />
3) NMRPipe Output Name: HSQC.fid<br />
4) Conversion Script Directory Option: working_dir<br />
5) Conversion Script Name: Convert_HSQC.csh<br />
6) Experiment Temperature (celsius) (from procpar/acqus file): 25<br />
7) Absolute Reference of DSS at experiment conditions (in MHz):<br />
<br />
Select parameter to change or [Return] to continue: 7<br />
<br />
WARNING: This overrides default behavior of AGNUS.<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Default behavior uses the DSS temperature referencing coefficients in the spectrometer table file.<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Leave this field blank or zero if you do not wish to override standard behavior.<br />
<br />
Absolute Frequency of DSS: 800.1899230<br />
</pre> <br />
This produces the following NMRPipe conversion file, which the user can then modify and subsequently run: <br />
<pre>Conversion script:<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
#!/bin/csh<br />
<br />
# This hsqcetf3gpsi conversion script was created using AGNuS 2.1.<br />
<br />
# Make the conversion directory if it does not exist (because var2pipe will not for 2D's)<br />
if (! -d ./pipe_conversion/ ) mkdir ./pipe_conversion/<br />
<br />
bruk2pipe -bad 0.0 -noaswap -DMX -decim 16 -dspfvs 12 -in ./ser \<br />
-xN 2048 -yN 256 \<br />
-xT 1024 -yT 128 \<br />
-xMODE DQD -yMODE Echo-AntiEcho \<br />
-xSW 11160.7142857143 -ySW 4865.4880848234 \<br />
-xOBS 800.19376489395 -yOBS 81.09146808039 \<br />
-xCAR 4.80122760796774 -yCAR 110.121740240004 \<br />
-xLAB 1H -yLAB 15N \<br />
-ndim 2 -aq2D States \<br />
-out ./pipe_conversion/HSQC.fid -verb -ov <br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
<br />
Known Nuclei: H,N<br />
</pre> <br />
A default NMRPipe processing script is then generated, which can again be modified by the user, and subsequently run: <br />
<pre>Full Processing script:<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
[1] #!/bin/csh<br />
[2] #<br />
[3] # This hsqcetf3gpsi processing script was created using AGNuS 2.1.<br />
[4] #<br />
[5] # 2D Processing Script.<br />
[6] <br />
[7] nmrPipe -in ./pipe_conversion/HSQC.fid \<br />
[8] | nmrPipe -fn SOL \<br />
[9] | nmrPipe -fn SP -off 0.45 -end 0.98 -pow 2 -c 0.5 \<br />
[10] | nmrPipe -fn ZF -auto \<br />
[11] | nmrPipe -fn FT \<br />
[12] | nmrPipe -fn PS -p0 -60 -p1 0 -di \<br />
[13] | nmrPipe -fn TP \<br />
[14] | nmrPipe -fn SP -off 0.45 -end 0.98 -pow 2 -c 0.5 \<br />
[15] | nmrPipe -fn ZF -auto \<br />
[16] | nmrPipe -fn FT \<br />
[17] | nmrPipe -fn PS -p0 -90 -p1 0 -di \<br />
[18] -verb -ov -out ./HSQC_SP.pipe<br />
[19]<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
<br />
</pre> <br />
Next the program launches NMRDraw (Figure 1).&nbsp; Here the user determines phase corrections in both dimensions, which are then applied to the processing script and the data is reprocessed. <br />
<br />
==== Figure 1: An NMRDraw Window for a <sup>1</sup>H,<sup>15</sup>N-HSQC ====<br />
<br />
[[Image:AGNuS NMRDraw.png|800px]]<br> <br />
<br />
Finally, the user converts to SPARKY&nbsp;or XEASY format, and proceeds to spectral analysis. <br />
<br />
<br> <br />
<br />
=== Processing a 3D Experiments Using AGNuS ===<br />
<br />
Processing higher dimensionality experiments using AGNuS is essentially the same process with a few minor differences. For example, for a 3D experiment, the user first produces F1F3 (xy) and F2F3 (xz) 2D&nbsp;planes which are phase corrected using NMRDraw.&nbsp; Then the 3D NMRPipe script is run to produce the 3D processed pipe file. <br />
<br />
<br> <br />
<br />
=== '''Processing a GFT&nbsp;Experiment Using AGNuS''' ===<br />
<br />
Here are some instructions for processing a GFT experiment: <br />
<br />
*Before running AGNuS, an appropriate pulse sequence table corresponding to the NMR data should be prepared. The table files for the most used NMR experiments are availble from AGNuS. AGNuS will automatically detects the procpar file, parses it, and selects the appropriate parameters from the procpar and the corresponding experiment description (pulse sequence table). Select <code>*MULTI</code> for GFT basic experiments process.<br />
<br />
'''Example of pulse sequence table for GFT_43D_CABCAcoNHN''' <br />
<pre>file_type&nbsp;: single_fid_table<br />
name&nbsp;: GFT_43D_CBCACAcoNHN<br />
nickname&nbsp;: HNcoCACB_GFT<br />
synonym&nbsp;: GFT_43D_CBCACAcoNHN_MULTI<br />
dimensions&nbsp;: [<br />
nuclei&nbsp;: H<br />
offset_parameter&nbsp;: tof<br />
acquisition_mode&nbsp;: Complex<br />
]<br />
dimensions&nbsp;: [<br />
nuclei&nbsp;: C<br />
offset_parameter&nbsp;: dof<br />
acquisition_mode&nbsp;: Complex<br />
]<br />
dimensions&nbsp;: [<br />
nuclei&nbsp;: N<br />
offset_parameter&nbsp;: dof2<br />
acquisition_mode&nbsp;: Complex<br />
]<br />
<br />
processing_defaults&nbsp;: [<br />
name&nbsp;: "Sine Bell Default"<br />
dimensions&nbsp;: [<br />
transformation&nbsp;: FT<br />
apodization&nbsp;: "SP -off 0.45 -end 0.98 -pow 2"<br />
first_point_correction&nbsp;: 0.5<br />
]<br />
dimensions&nbsp;: [<br />
transformation&nbsp;: FT<br />
apodization&nbsp;: "SP -off 0.45 -end 0.98 -pow 1"<br />
first_point_correction&nbsp;: 1.0<br />
]<br />
dimensions&nbsp;: [<br />
transformation&nbsp;: "FT -neg"<br />
apodization&nbsp;: "SP -off 0.45 -end 0.98 -pow 1"<br />
first_point_correction&nbsp;: 0.5<br />
]<br />
xy_plane&nbsp;: 002<br />
]</pre> <br />
*To run AGNuS, simply go to the directory only contains the Varian fid and procpar (parameter) file; type <code>agnus</code> and follow the instructions prompted by AGNuS. Type <code>agnus -help</code> for help. <br />
*#Select <code>*MULTI</code> for GFT basic experiments process (optional, AGNuS will go to the next step if non-GFT data if identified) <br />
*#Selecting the spectrometer <br />
*#Select the basic information about where files are located, name of conversion scripts, and names of converted files. <br />
*#Run the conversion script provided by AGNuS to convert NMR data format to Pipe format. Modify the conversion script is necessary. <br />
*#Run the process scripts (3D and XY, XZ planes process scripts for 3D data) provided by AGNuS. Modify the Buffalo.NMRPipe process parameters such as phase values if necessary. <br />
*##AGNuS will lauch NMRDraw to let the user to check the process data and get the phase values. <br />
*##Use <code>CTRL-C</code> to quit AGNuS after phasing the spectrum with NMRDraw <br />
*##Rerun AGNuS and manually type in or correct the phase when come to the process scripts change step. Modify the base line parameter as well if necessary <br />
*##Run the final process after all parameters be carefully reviewed. <br />
*#Run the UCSF conversion and cleanup script if use SPARKY for NMR data analysis. Run [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/SPSCAN SPSCAN] to conver Buffalo.NMRPipe format data to [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/XEASY XEASY] format data if use [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/XEASY XEASY].<br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=AGNuS/AutoProc&diff=3847
AGNuS/AutoProc
2011-10-05T15:56:50Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
[http://www-nmr.cabm.rutgers.edu/software/autoproc.htm Autoproc/AGNuS] is a suite of programs for the automatic generation of scripts for multidimensional protein NMR data processing and format conversion. AGNuS automatically generates NMRPipe scripts for processing multidimensional Varian or Bruker NMR experiments and provides functions for chemical referencing.&nbsp; AGNuS calls information&nbsp; for specific experiments and spectrometers stored in table files, which can be generated/modified by users with permission.&nbsp; AGNuS is also capable of processing reduced dimensionality/GFT experiments.&nbsp; <br />
<br />
The current version of the program is AutoProc 2.0.&nbsp; Please check the [[Media:AGNuS_help.jpg|documentation]] and examples provided with the software for addtional information.<br> <br />
<br />
== '''How to Use AGNuS''' ==<br />
<br />
=== Processing a 2D <sup>1</sup>H,<sup>15</sup>N-HSQC Using AGNuS ===<br />
<br />
AGNuS parses procpar (Varian) and acqu* (Bruker) files for spectral parameters.&nbsp; The program consults a pulse sequence table specific to the sequence used in the experiment.&nbsp; For example, this is the table file for the standard Bruker 2D <sup>1</sup>H,<sup>15</sup>N-HSQC, hsqcetf3gpsi.&nbsp; If the sequence table file is missing from the Pulse_Sequences directory in AGNuS, the program will throw an error and the user must create one (best to clone one from an similar one already in the directory).<br> <br />
<pre># This is a Bruker experiment and only a table for testing.<br />
<br />
file_type&nbsp;: single_fid_table<br />
name&nbsp;: hsqcetf3gpsi<br />
nickname&nbsp;: HSQC<br />
dimensions&nbsp;: [<br />
nuclei&nbsp;: H<br />
offset_parameter&nbsp;: O1_1<br />
acquisition_mode&nbsp;: DQD<br />
]<br />
dimensions&nbsp;: [<br />
nuclei&nbsp;: N<br />
offset_parameter&nbsp;: O1_2<br />
acquisition_mode&nbsp;: Echo-AntiEcho<br />
]<br />
<br />
processing_defaults&nbsp;: [<br />
&nbsp; name&nbsp;: "Sine Bell Default"<br />
&nbsp; suffix&nbsp;: _SP<br />
&nbsp; dimensions&nbsp;:&nbsp; [<br />
&nbsp;&nbsp;&nbsp; transformation&nbsp;: FT<br />
&nbsp;&nbsp;&nbsp; apodization&nbsp;: "SP -off 0.45 -end 0.98 -pow 2"<br />
&nbsp;&nbsp;&nbsp; first_point_correction&nbsp;: 0.5<br />
&nbsp;&nbsp;&nbsp; phase0&nbsp;: -60<br />
&nbsp;&nbsp;&nbsp; ]<br />
&nbsp; dimensions&nbsp;:&nbsp; [<br />
&nbsp;&nbsp;&nbsp; transformation&nbsp;: FT<br />
&nbsp;&nbsp;&nbsp; apodization&nbsp;: "SP -off 0.45 -end 0.98 -pow 2"<br />
&nbsp;&nbsp;&nbsp; first_point_correction&nbsp;: 0.5<br />
&nbsp;&nbsp;&nbsp; phase0&nbsp;: -90<br />
&nbsp;&nbsp;&nbsp; ]<br />
&nbsp; ]<br />
<br />
</pre> <br />
Start autoproc and enter a spectrometer, then a DSS value for referencing.&nbsp; This is the most optimal way to reference a spectrum.&nbsp; We reference to internal DSS in all of our production work. <br />
<br />
In a typical data production run collection we start with a 1D <sup>1</sup>H NMR spectrum and obtain the absolute resonance frequency of DSS under the conditions used for the NMR experiments.&nbsp; This value is then used <br />
<br />
in the subsequent AutoProc processing of all fids from the data collection, as described below (option 7).<br />
<pre>AGNuS 2.1<br />
&nbsp; Type "agnus -help" for help<br />
&nbsp; Documentation is located at URL file:/farm/software/NewAutoProc2.0/help_files/index.html<br />
<br />
<br />
WARNING *** SW_h (2000) does not equal SW * SFO1 (4865.4880848234). Setting SW_h = SW * SFO1.<br />
<br />
Experiment hsqcetf3gpsi selected.<br />
<br />
<br />
** Spectrometer Selection Menu **<br />
<br />
1) CABM_Bruker_500<br />
2) CABM_Bruker_600_100306<br />
3) CABM_Varian_500<br />
4) CABM_Varian_500_060805_20C<br />
5) CABM_Varian_600<br />
6) NYSBC_800_Conventional<br />
7) PNNL_Baker_600<br />
8) PNNL_Glacier_600<br />
9) PNNL_Varian_500<br />
10) PNNL_Varian_750<br />
11) PNNL_Varian_800<br />
12) SUNY_Buffalo_Varian_600<br />
13) SUNY_Buffalo_Varian_750<br />
14) Z_500_No_Reference<br />
15) Z_600_No_Reference<br />
16) Z_750_No_Reference<br />
17) Z_800_No_Reference<br />
<br />
Spectrometer [1]: 17<br />
<br />
** Conversion Parameter Selection Menu **<br />
<br />
-- Current Directory: /farm/data/jma/JIM_NESG_PROJECTS/PfR193A_PROJECT/FIDs/B800/PfR193A.019_NC5_062509/NHhsqc_largeSW1_exp4<br />
-- Input Directory: ./<br />
1) Input Data File (FID) Name: ser<br />
2) Working Directory: ./<br />
-- Conversion Subdirectory: pipe_conversion/<br />
3) NMRPipe Output Name: HSQC.fid<br />
4) Conversion Script Directory Option: working_dir<br />
5) Conversion Script Name: Convert_HSQC.csh<br />
6) Experiment Temperature (celsius) (from procpar/acqus file): 25<br />
7) Absolute Reference of DSS at experiment conditions (in MHz):<br />
<br />
Select parameter to change or [Return] to continue: 7<br />
<br />
WARNING: This overrides default behavior of AGNUS.<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Default behavior uses the DSS temperature referencing coefficients in the spectrometer table file.<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Leave this field blank or zero if you do not wish to override standard behavior.<br />
<br />
Absolute Frequency of DSS: 800.1899230<br />
</pre> <br />
This produces the following NMRPipe conversion file, which the user can then modify and subsequently run: <br />
<pre>Conversion script:<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
#!/bin/csh<br />
<br />
# This hsqcetf3gpsi conversion script was created using AGNuS 2.1.<br />
<br />
# Make the conversion directory if it does not exist (because var2pipe will not for 2D's)<br />
if (! -d ./pipe_conversion/ ) mkdir ./pipe_conversion/<br />
<br />
bruk2pipe -bad 0.0 -noaswap -DMX -decim 16 -dspfvs 12 -in ./ser \<br />
-xN 2048 -yN 256 \<br />
-xT 1024 -yT 128 \<br />
-xMODE DQD -yMODE Echo-AntiEcho \<br />
-xSW 11160.7142857143 -ySW 4865.4880848234 \<br />
-xOBS 800.19376489395 -yOBS 81.09146808039 \<br />
-xCAR 4.80122760796774 -yCAR 110.121740240004 \<br />
-xLAB 1H -yLAB 15N \<br />
-ndim 2 -aq2D States \<br />
-out ./pipe_conversion/HSQC.fid -verb -ov <br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
<br />
Known Nuclei: H,N<br />
</pre> <br />
A default NMRPipe processing script is then generated, which can again be modified by the user, and subsequently run: <br />
<pre>Full Processing script:<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
[1] #!/bin/csh<br />
[2] #<br />
[3] # This hsqcetf3gpsi processing script was created using AGNuS 2.1.<br />
[4] #<br />
[5] # 2D Processing Script.<br />
[6] <br />
[7] nmrPipe -in ./pipe_conversion/HSQC.fid \<br />
[8] | nmrPipe -fn SOL \<br />
[9] | nmrPipe -fn SP -off 0.45 -end 0.98 -pow 2 -c 0.5 \<br />
[10] | nmrPipe -fn ZF -auto \<br />
[11] | nmrPipe -fn FT \<br />
[12] | nmrPipe -fn PS -p0 -60 -p1 0 -di \<br />
[13] | nmrPipe -fn TP \<br />
[14] | nmrPipe -fn SP -off 0.45 -end 0.98 -pow 2 -c 0.5 \<br />
[15] | nmrPipe -fn ZF -auto \<br />
[16] | nmrPipe -fn FT \<br />
[17] | nmrPipe -fn PS -p0 -90 -p1 0 -di \<br />
[18] -verb -ov -out ./HSQC_SP.pipe<br />
[19]<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
<br />
</pre> <br />
Next the program launches NMRDraw (Figure 1).&nbsp; Here the user determines phase corrections in both dimensions, which are then applied to the processing script and the data is reprocessed. <br />
<br />
==== Figure 1: An NMRDraw Window for a <sup>1</sup>H,<sup>15</sup>N-HSQC ====<br />
<br />
[[Image:AGNuS NMRDraw.png|800px]]<br> <br />
<br />
Finally, the user converts to SPARKY&nbsp;or XEASY format, and proceeds to spectral analysis. <br />
<br />
<br> <br />
<br />
=== Processing a 3D Experiments Using AGNuS ===<br />
<br />
Processing higher dimensionality experiments using AGNuS is essentially the same process with a few minor differences. For example, for a 3D experiment, the user first produces F1F3 (xy) and F2F3 (xz) 2D&nbsp;planes which are phase corrected using NMRDraw.&nbsp; Then the 3D NMRPipe script is run to produce the 3D processed pipe file. <br />
<br />
<br> <br />
<br />
=== '''Processing a GFT&nbsp;Experiment Using AGNuS''' ===<br />
<br />
Here are some instructions for processing a GFT experiment: <br />
<br />
*Before running AGNuS, an appropriate pulse sequence table corresponding to the NMR data should be prepared. The table files for the most used NMR experiments are availble from AGNuS. AGNuS will automatically detects the procpar file, parses it, and selects the appropriate parameters from the procpar and the corresponding experiment description (pulse sequence table). Select <code>*MULTI</code> for GFT basic experiments process.<br />
<br />
'''Example of pulse sequence table for GFT_43D_CABCAcoNHN''' <br />
<pre>file_type&nbsp;: single_fid_table<br />
name&nbsp;: GFT_43D_CBCACAcoNHN<br />
nickname&nbsp;: HNcoCACB_GFT<br />
synonym&nbsp;: GFT_43D_CBCACAcoNHN_MULTI<br />
dimensions&nbsp;: [<br />
nuclei&nbsp;: H<br />
offset_parameter&nbsp;: tof<br />
acquisition_mode&nbsp;: Complex<br />
]<br />
dimensions&nbsp;: [<br />
nuclei&nbsp;: C<br />
offset_parameter&nbsp;: dof<br />
acquisition_mode&nbsp;: Complex<br />
]<br />
dimensions&nbsp;: [<br />
nuclei&nbsp;: N<br />
offset_parameter&nbsp;: dof2<br />
acquisition_mode&nbsp;: Complex<br />
]<br />
<br />
processing_defaults&nbsp;: [<br />
name&nbsp;: "Sine Bell Default"<br />
dimensions&nbsp;: [<br />
transformation&nbsp;: FT<br />
apodization&nbsp;: "SP -off 0.45 -end 0.98 -pow 2"<br />
first_point_correction&nbsp;: 0.5<br />
]<br />
dimensions&nbsp;: [<br />
transformation&nbsp;: FT<br />
apodization&nbsp;: "SP -off 0.45 -end 0.98 -pow 1"<br />
first_point_correction&nbsp;: 1.0<br />
]<br />
dimensions&nbsp;: [<br />
transformation&nbsp;: "FT -neg"<br />
apodization&nbsp;: "SP -off 0.45 -end 0.98 -pow 1"<br />
first_point_correction&nbsp;: 0.5<br />
]<br />
xy_plane&nbsp;: 002<br />
]</pre> <br />
*To run AGNuS, simply go to the directory only contains the Varian fid and procpar (parameter) file; type <code>agnus</code> and follow the instructions prompted by AGNuS. Type <code>agnus -help</code> for help. <br />
*#Select <code>*MULTI</code> for GFT basic experiments process (optional, AGNuS will go to the next step if non-GFT data if identified) <br />
*#Selecting the spectrometer <br />
*#Select the basic information about where files are located, name of conversion scripts, and names of converted files. <br />
*#Run the conversion script provided by AGNuS to convert NMR data format to Pipe format. Modify the conversion script is necessary. <br />
*#Run the process scripts (3D and XY, XZ planes process scripts for 3D data) provided by AGNuS. Modify the Buffalo.NMRPipe process parameters such as phase values if necessary. <br />
*##AGNuS will lauch NMRDraw to let the user to check the process data and get the phase values. <br />
*##Use <code>CTRL-C</code> to quit AGNuS after phasing the spectrum with NMRDraw <br />
*##Rerun AGNuS and manually type in or correct the phase when come to the process scripts change step. Modify the base line parameter as well if necessary <br />
*##Run the final process after all parameters be carefully reviewed. <br />
*#Run the UCSF conversion and cleanup script if use SPARKY for NMR data analysis. Run [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/SPSCAN SPSCAN] to conver Buffalo.NMRPipe format data to [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/XEASY XEASY] format data if use [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/XEASY XEASY].<br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Structure_Refinement_Using_CNS_Energy_Minimization_With_Explicit_Water&diff=3846
Structure Refinement Using CNS Energy Minimization With Explicit Water
2011-10-05T15:28:07Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
Crystallography &amp; NMR System (CNS) is a program has been designed to provide a flexible multi-level hierachical approach for the most commonly used algorithms in macromolecular structure determination [1]. Functions include heavy atom searching, experimental phasing (including MAD and MIR), density modification, crystallographic refinement with maximum likelihood targets, and NMR structure calculation using NOEs, J-coupling, chemical shift, and dipolar coupling data. <br />
<br />
The latest version is 1.21. Please check the website [http://cns-online.org/ http://cns-online.org] for additional information. <br />
<br />
== '''CNS Refinement Protocol''' ==<br />
<br />
The final structure from CYANA should be refined using CNS energy minimization with explicit water before PDB deposition. <br />
<br />
The common input files are: <br />
<br />
*PDB coordinates <br />
*NOE constraints <br />
*Dihedral angle constraints <br />
*Hydrogen bond constraints<br />
<br />
==== '''Converting input files from CYANA to XPLOR/CNS''' ====<br />
<br />
If the final structure calculation was performed with CYANA 2.1, then the constraint and coordinate files must be first converted to XPLOR/CNS format. <br />
<br />
==== '''Using cyana2cns.cya script''' ====<br />
<br />
This procedure can be used to prepare input files to use with the WaterRefCNS script (see below). <br />
<br />
#Download [[Media:Cyana2cns.cya|cyana2cns.cya]] into directory with the final structure calculation. <br />
#Modify the <tt>read pdb</tt>, <tt>read upl</tt> and <tt>read aco</tt> lines if necessary. <br />
#Start CYANA 2.1 and run <tt>cyana2cns.cya</tt>.<br />
<br />
*All conformers will be stored in a single PDB file <tt>KKK.pdb</tt>. If you plan run water bath refinement manually, use the <tt>p2X</tt> program instead. <br />
*The resulting NOE constraint file <tt>KKK_noe.tbl</tt> will have all lower limits set to 0. If want them to be set according to VdW radii, use the <tt>r2X</tt> program instead. Experience shows that there is no significant effect on geometry and quality scores as reported by PSVS.<br />
<br />
==== '''Using programs p2X and r2X and d2X''' ====<br />
<br />
These programs were written by Alex Lemak at the University of Toronto. They are typically used to prepare input files for manual CNS water bath refinement. <br />
<br />
Go to <tt>~1_projects/targetID/structure/cns/</tt> directory and <tt>conversion_scripts</tt> sub directory, convert required input files to CNS format from CYANA format by using macro <tt>mkfil</tt>:&nbsp; <br />
<pre> p2X cyana2.pdb pref <br />
r2X found-c.upl noeupl.tbl <br />
r2X gr-hbonds.upl hbonds.tbl<br />
cat noeupl.tbl hbonds.tbl &gt; noe.tbl <br />
d2X found-c.aco aco.tbl <br />
</pre> <br />
*<tt>p2X splits conformers from a pdb file generated by CYANA 2.1 into individual pdb files (one file per conformer) and converts atom names to CNS format. The second argument "pref"</tt> specifies a common prefix name for the output pdb files. It should be no more than 4 characters long. <br />
*<tt>r2X</tt> converts distance constraints (noe/hbond) from CYANA 2.1 format to CNS format. The lower limits in the resulting constraint file are set according to VdW radii. <br />
**Distance constraints obtained from CYANA macro <tt>caliba</tt> or <tt>calibration</tt> can be used as input files. <br />
**Distance constraints obtained from CYANA macro <tt>peak calibrate</tt> should have the pseudo-atom correction run before used as input files, run following commands under CYANA: <br> <pre>read upl final.upl<br />
</pre><br />
<br />
distance correct write upl final_corrected.upl This, however, will not add corrections for multiplicity! <br />
<br />
*<tt>d2X</tt>, to run: <tt>d2X cyana2.aco cns.tbl</tt> This will convert angle constraints from CYANA format to CNS format. If you don't have any dihedral angle constrains, create an empty file.<br />
<br />
Both p2X and r2X require a translation table file [[Media:AtomtransC.tbl|atomtransC.tbl]]. <br />
<br />
If have used simplified pseudoatom names (H* instead of Q*) with option pseudo=2 in CYANA 2.1, you may first want to them before running p2X and r2X:<br> <br />
<pre>pseudo=0<br />
read upl noe.upl<br />
read pdb cyana2.pdb<br />
write upl out.upl<br />
write pdb out.pdb all<br />
</pre> <br />
Note that pseudo=0 should be set '''before''' loading PDB and constraint files. <br />
<br />
==== '''Conversion from DYANA''' ====<br />
<br />
Use CYANA macro <tt>MigrateFromDyanaCyana1.cya</tt> to convert of data files from standard CYANA1.x or DYANA nomenclature to the standard IUPAC nomenclature used by CYANA, then do then same thing as above. <br> <br />
<pre> translate dyana # use Cyana 1.x/Dyana nomenclature<br />
read seq demo-dyana.seq # read sequence from Cyana 1.x/Dyana<br />
read upl demo-dyana.upl unknown=warn # read upper distance limits from Cyana 1.x/Dyana<br />
read aco demo-dyana.aco unknown=warn # read angle restraints from Cyana 1.x/Dyana<br />
translate off # return to standard (IUPAC) nomenclature<br />
write demo-cyana.seq # save sequence in standard Cyana format<br />
write upl demo-cyana.upl # save upper distance limits in standard Cyana format<br />
write aco demo-cyana.aco # save angle restraints in standard Cyana format <br />
</pre> <br />
<br> <br />
<br />
=== '''Running CNS using WaterRefCNS script at CABM''' ===<br />
<br />
The WaterRefCNS script, developed at Rutgers University by Roberto Tejero, performs many of the above CNS water refinement protocols in an easy to use script with many user controled options. <br />
<br />
To see a list of options type the command: <br />
<pre> WaterRefCNS<br />
</pre> <br />
You will see a list of options and how the program is used. <br />
<pre> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
WaterRefCN -- a tool to launch structure refinement with water<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
<br />
Usage: /farm/software/WaterRefinement_cns/WaterRefCNS -na NamProt -que [PBS|No] -pro [xpl|cns]<br />
<br />
** Full set of options [not all are needed for a perfect run, use defaults]<br />
<br />
-na NamProt Name of the protein NOTE: this is mandatory, no default value <br />
-qu [PBS|NO] Que system to use, one of PBS or NOQUE def NOQUE<br />
-pr [cns|xpl] Protocol to use, X-POR, CNS def cns <br />
-av [center|sum] average for the distance restraints (for CNS, XPLOR) def sum <br />
-ci n1,n2 CIS pep info (PRO res num) in comma separated list, i.e 56,89 <br />
-ss n1-n2,n3-n4 S-S bridge info in comma separated, dash-separated pairs, i.e. 2-24,30-40 <br />
-he Display this help message<br />
-hisd n1,n2 HISD pep info (HIS res num) in comma separated list, i.e 12,43<br />
-hise n1,n2 HISE pep info (HIS res num) in comma separated list, i.e 25,67<br />
-heat N number of cycles in heating stage, default is 200 <br />
-hot N number of cycles in HOT stage, default is 1000 <br />
-cool N number of cycles for cooling stage, default is 100 <br />
-sc N Scale all terms (noe, dihe, hbond) by xN times def 1 <br />
-par string Choice of nonbonded params, one of OPLSX|PARAM19|PARMALLH6|PROLSQ|CONTACT<br />
default is OPLSX<br />
-seed &lt;int&gt; Seed for random number generator, default 31415 <br />
<br />
Examples:<br />
/farm/software/WaterRefinement_cns/WaterRefCNS -na SR358 -que no -pr cns -ci 22 <br />
<br />
</pre> <br />
Given a protein name KKK, here are the input files required to run WaterRefCNS: <br />
<br />
*&nbsp; pdb coordinates (in a single file) prepared in Xplor format using PdbStat.&nbsp; The name of the file should be:&nbsp; <tt>KKK.pdb</tt> <br />
*&nbsp; an noe.tbl file in CNS format.&nbsp; You can convert CYANA a&nbsp;final.upl file using PdbStat.&nbsp; We typically add 10-15% to the upper limit and make the lower limit vdw.&nbsp; The name of this file should be:&nbsp; <tt>KKK_noe.tbl</tt> <br />
*&nbsp; a dihe.tbl file in CNS&nbsp;format.&nbsp; Again, you can convert a CYANA .aco file using PdbStat.&nbsp; The name of this file should be:&nbsp; <tt>KKK_dihe.tbl</tt> <br />
*&nbsp; an hbond.tbl file in CNS format (optional).&nbsp; The name of this file should be:&nbsp; <tt>KKK_bhond.tbl</tt><br />
<br />
<br> <br />
<br />
In order to run WaterRefCNS on the CABM-NMR cluster: <br />
<br />
#Log into hummer or master3 <br />
#go to the directory with the above 4 files <br />
#type:<br />
<pre> /farm/software/WaterRefinement_cns/WaterRefCNS -na KKK -que PBS</pre> <br />
and that's it.&nbsp; If you have some CIS residue you add <tt>-ci</tt> NUMRES to the options above. If you want to have better vdw violation, you can add <tt>-par PARAM19</tt> to the options above. <br />
<br />
'''Future work:''' <br />
<br />
Currently, the WaterRefCNS script does not support refinement with RDC's.&nbsp; <br />
<br />
<br> <br />
<br />
=== '''WaterRefCNS at SUNY Buffalo''' ===<br />
<br />
The CABM water refinement package was customized to run on computers at UB: <br />
<br />
*Fixed a bug in <tt>WaterRefCNS</tt> so that the assembled refined PDB structure file has a <tt>.pdb</tt> extension. <br />
*PBS queue submission code in <tt>WaterRefCNS</tt> modified for use on U2 cluster <br />
*Added <tt>-hisd</tt> and <tt>-hise</tt> options to use neutral histidine isoprotomers. This required modification of <tt>WaterRefCNS</tt>, <tt>cns_refine_h2o.inp</tt> and <tt>generate_h2o.inp</tt>. <br />
*Fixed a bug in the protein topology file <tt>topallhdg5.3.pro</tt>. The line <tt>atom CD1 type=CR1E charge=0.130 end</tt> in the HISE patch section was removed. <br />
*Fixed a bug that caused the order of conformers to be changed in the output PDB file by replacing the line <tt>$WATREFLIB/Agrupa *.pdb &gt; All_${Name}_cns.pdb</tt> with <tt>$WATREFLIB/Agrupa `ls resa_?.pdb&nbsp;; ls resa_??.pdb` &gt; All_${Name}_cns.pdb</tt> in the <tt>WaterRefCNS</tt> script.<br />
<br />
The modified package is installed in <tt>/nsm/chem/cen2/HTP2/3_src/WaterRefinement_cns</tt> on the local server and in <tt>/san/projects1/szypersk/src/WaterRefinement_cns</tt> on the U2 Linux cluster. Make sure that this directories is included in your path on <tt>spins*</tt> workstations and U2, respectively. <br />
<br />
For reference, the download location is here: [[Media:WaterRefinement_UB.tar.Z|WaterRefinement_UB.tar.Z]] <br />
<br />
The required files are: <br />
<br />
*PDB coordinates (with atom names in XPLOR/CNS format) <br />
*NOE constraints (in XPLOR/CNS format) <br />
*Dihedral angle constraints (in XPLOR/CNS format) <br />
*Hydrogen bond constraints (in XPLOR/CNS format)<br />
<br />
Assuming that your coordinate file is called <tt>KKK.pdb</tt>, the constraint files should be named <tt>KKK_noe.tbl</tt>, <tt>KKK_dihe.tbl</tt> and <tt>KKK_hbond.tbl</tt>. If you don't have dihedral angle and/or hydrogen bond constraints, then create the corresponding empty files. <br />
<br />
You may need to add a line like <tt>nassign=1000</tt> at the top of the constraint files. The value should be equal to or greater than the number of constraints in the file. <br />
<br />
WaterRefCNS assumes "sum of r<sup>-6</sup>" averaging by default. When using "center"-averaged UPLs (e.g. from <tt>caliba</tt>) add <tt>-av center</tt> when starting WaterRefCNS. For information on averaging conventions for calibration in CYANA see [[NOE Calibration Using CYANA|NOE Calibration in CYANA]] <br />
<br />
It is recommended to run it on U2 Linux cluster. Examples: <br />
<br />
#<pre>WaterRefCNS -na KKK -que PBS</pre> <br />
#<pre>WaterRefCNS -na KKK -que PBS -ci 21,49</pre>residues 21 and 49 are cis-Pro <br />
#<pre>WaterRefCNS -na KKK -que PBS -hise 40,82 -hisd 32,65</pre>residues 40 and 82 are ε-protonated neutral His <br> residues 32 and 65 are δ-protonated neutral His <br />
#<pre>WaterRefCNS -na KKK -que PBS -av center </pre>for use with center-averaged calibration<br />
<br />
To run on <tt>spins*</tt> Linux workstations without queue system use <tt>-que NO</tt> option instead of <tt>-que PBS</tt> option. The default is no queue system, so you can omit the <tt>-que</tt> option altogether. <br />
<br />
==== '''Running WaterRefCNS''' ====<br />
<br />
#Convert the coordinate and constraint files as described above. <br />
#Copy the converted files into a CNS working directory on the U2 cluster or a workstation (e.g. <tt>structure/cns/calc1</tt>). <br />
#Run the <tt>WaterRefCNS</tt> with the proper arguments. <br />
#If the calculation was successful, the refined coordinate file will be stored in the <tt>refinedPDB</tt> subdirectory. Check the <tt>BeSureToREADME</tt> for details.<br />
<br />
=== '''Running CNS "manually"''' ===<br />
<br />
Go to <tt>~1_projects/targetID/structure/cns/</tt> directory and <tt>cns_scripts</tt> sub directory, follow the procedures as describe below. <br />
<br />
'''1.&nbsp; Generating MTF topology file from a PDB file. '''<br>&nbsp;&nbsp;&nbsp; modify the header of the input file <tt>generate_mtf.inp</tt> and then run <tt>cns &lt; generate_mtf.inp &gt; mtf.log</tt>. <br />
<br />
*Modify input files path and name base on individual target as follows:<br />
<pre> ...<br />
{in} pdb_file="/san/user/gliu2/u2/cns/cns/convertion_scripts/ufc1_1.pdb";<br />
{in} param_file="/san/user/gliu2/u2/cns/cns/cns_scripts/parallhdg5.3C.pro";<br />
{in} topol_file="/san/user/gliu2/u2/cns/cns/cns_scripts/topallhdg5.3.pro";<br />
{in} plink_file="/san/user/gliu2/u2/cns/cns/cns_scripts/protein.link";<br />
{out} struct_file="ufc1_1.mtf"; <br />
...<br />
</pre> <br />
*Requires these files: <br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file <br />
*[[Media:Protein.link|protein.link]]: CNS peptide linkage definition file <br />
*For <tt>cis-Proline</tt>, define the residue Id of the residue prior to <tt>cis-Proline</tt> by additional lines in <tt>generate_mtf.inp</tt> before the WRITE command<br />
<pre> patch cisp<br />
reference=nil=( resid 90 )<br />
end <br />
</pre> <br />
*If you get an error message on the atom type, you also can try&nbsp;<br />
<pre> patch cipp<br />
reference=nil=( resid 90 )<br />
end </pre> <br />
*For neutral Nε- and Nδ-protonated histidines add these lines in <tt>generate_mtf.inp</tt> before the WRITE command<br />
<pre> patch hise<br />
reference=nil=( resid 40 )<br />
end<br />
patch hisd<br />
reference=nil=( resid 65 )<br />
end </pre> <br />
*For dimer refinement, add a line containing only <tt>TER</tt> between two monomer unit coordinates in the PDB conformer that is defined in <tt>generate_mtf_cis.inp</tt> as input file.<br />
<br />
'''<br>''' <br />
<br />
'''2.&nbsp; Rebuilding hydrogen atom positions for each structure. '''<br>&nbsp;&nbsp;&nbsp; Modify input file <tt>rebuild.inp</tt> and then run <tt>cns &lt; rebuild.inp &gt; rebuild.log</tt> <br />
<br />
*Modify input files path and name base on induvidual target as follows:&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;<br />
<pre> ...<br />
evaluate ($mtf_file="ufc1_1.mtf")<br />
evaluate ($pdbname_in="../convertion_scripts/ufc1")<br />
evaluate ($pdbname_out="ufc1_rb")<br />
evaluate ($number_of_struct= 20 )<br />
...<br />
evaluate ($topol_p_file="/san/user/gliu2/u2/cns/cns/cns_scripts/topallhdg5.3.pro")<br />
evaluate ($param_p_file="/san/user/gliu2/u2/cns/cns/cns_scripts/parallhdg5.3C.pro")<br />
...<br />
</pre> <br />
*Requires these files: <br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file<br />
<br />
'''3. Refining structures with explicit water.''' <br />
<br />
*Running <tt>CNS</tt> on workstation with single processor, modify input file <tt>re_h2oc.inp</tt> and then run <tt>cns &lt; re_h2oc.inp &gt; h2o.log</tt> <br />
*Modify input files path and name base on induvidual target as follows:<br />
<pre> ...<br />
evaluate ($mtf_file="ufc1_1.mtf")<br />
evaluate ($noe_file="../noe.tbl")<br />
evaluate ($dihe_file="../aco.tbl")<br />
evaluate ($hb_file="../hbond.tbl")<br />
evaluate ($pdbname_in="ufc1_rb")<br />
evaluate ($pdbname_out="ufc1_ref")<br />
evaluate ($number_of_struct= 20 )<br />
...</pre> <br />
*Modify the weight of energy contribution part if necessary, e.g. use 2x to 10x of the following scales:<br />
<pre> scale ambi 50<br />
scale dist 50<br />
scale hbond 50<br />
end<br />
restraints dihedral<br />
scale=200<br />
end</pre> <br />
*Requires <br />
**[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
**[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file <br />
**[[Media:Topallhdg5.3.sol|topallhdg5.3.sol]]: CNS solvent topology file <br />
**[[Media:Parallhdg5.3.sol|parallhdg5.3.sol]]: CNS solvent parameter file <br />
**[[Media:Boxtyp20.pdb|boxtyp20.pdb]]: Water box <br />
**[[Media:Generate_water.cns|generate_water.cns]]: CNS protein solvation script <br />
**[[Media:Print_coorheader.cns|print_coorheader.cns]]: CNS script <br />
*Run CNS on workstation with multiple processors. <br> CNS will refine each conformer independently and in parallel, it will be much faster if 20 conformer were refined in parallal at u2.ccr.buffalo.edu.<br />
<br />
&nbsp;&nbsp;&nbsp; 1.&nbsp; Run cns_convertion as descibed above &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp; 2.&nbsp; Go to cns_scripts directory, modify input file <tt>generate_mtf_cis.inp</tt> and then run =cns &lt; generate_mtf_cis.inp = as described above &nbsp;&nbsp;&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp; 3.&nbsp; Run macro <tt>getfil</tt> <br />
<pre> cp ../convertion_scripts/noe.tbl .<br />
cp ../convertion_scripts/hbonds.tbl .<br />
cp ../convertion_scripts/aco.tbl .<br />
cp ufc1_1.mtf com/.</pre> <br />
&nbsp;&nbsp;&nbsp; 4.&nbsp; Modify input file <tt>rebuild.inp</tt> and then run <tt>cns &lt; rebuild.inp</tt> as described above. <br />
<br />
&nbsp;&nbsp;&nbsp; 5.&nbsp; Modify input file <tt>re_h2oc.inp</tt> as described above, remember to set <tt>$number_of_struct = 20</tt>. <br />
<br />
&nbsp;&nbsp;&nbsp; 6.&nbsp; Run macro <tt>subaba</tt> <br />
<br />
<code></code> <br />
<pre> #bash<br />
for i in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20<br />
do<br />
mkdir ref$i<br />
cd ref$i<br />
cp ../com/* .<br />
cp ../jan24/ufc1_rb_$i.pdb ufc1_rb_1.pdb<br />
qsub cns.sc<br />
cd ..<br />
<br />
donedone</pre> <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; where <tt>cns.sc</tt> is a PBS macro used to submit CNS job by using PBS: <br />
<pre> #!/bin/csh<br />
#PBS -m e<br />
#PBS -q short_c<br />
#PBS -l nodes=1:GM:ppn=1<br />
#PBS -l walltime=00:56:00<br />
#PBS -o out1<br />
#PBS -j oe<br />
#PBS -N clean1<br />
cd $PBS_O_WORKDIR<br />
echo "working directory = "$PBS_O_WORKDIR<br />
set NN = `cat $PBS_NODEFILE | wc -l`<br />
echo "NN = "$NN<br />
# Run Job<br />
cns &lt; re_h2oc.inp<br />
echo "ALL Done!"</pre> <br />
&nbsp;&nbsp;&nbsp; 7.&nbsp; Run <tt>getpdb</tt> to collect refined structures. <br />
<pre> #bash<br />
for i in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20<br />
do<br />
cd ref$i<br />
cp ufc1_ref_1.pdb ../pdb/ufc1_ref_$i.pdb<br />
cp ufc1_ref_1.vio ../pdb/ufc1_ref_$i.vio<br />
cd ..<br />
done</pre> <br />
&nbsp;&nbsp;&nbsp; 8. Run script [[Media:Agrupa.txt|Agrupa]] to put all 20 conformers into a single PDB file.<tt></tt> <br />
<pre> Agrupa ufc1_ref*.pdb &gt; ufc1all.pdb</pre> <br />
=== '''Running CNS for Proteins with Metal Ions''' ===<br />
<br />
The CNS refinement for proteins with metal ions can also be performed with the new WaterrefCNS script, using the appropriate metal ion nomenclature in both the pdb coordinates and distance constraints. <br />
<br />
For manual CNS refinement please do the refinement as following: <br />
<br />
1.&nbsp; Set environment for CNS1.1 by run source /farm/users/gliu/alias.cns <br />
<pre> alias cns1 /farm/software/cns/cns_solve_1.1/intel-i686-linux_g77/bin/cns<br />
setenv CNS_TOPPAR /farm/data/gliu/cns1/</pre> <br />
In addition to the topology and parameter files, the metal ion parameter file <tt>ion.top</tt> is required. An example can be found in "/farm/users/gliu/projects/cns_cuttha_cis" with all required input files. 2.&nbsp; Prepared required files as described above (final.tbl, final cns format PDB files and put in xplorPDB dir with name as sa_#.pdb) except the PDB file should include the metal ions with format according to [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/CNS CNS] library <tt>ion.top</tt>. cp sa_1.pdb as&nbsp; template.pdb, input files for creating mtf file Note that alignment is important. eg: <br />
<pre>ATOM 1249 OT2 ALA 83 69.296 13.232 5.744 1.00 0.00<br />
ATOM 1250 ZN+2 ZN2 150 63.086 13.789 -10.407 1.00 0.00 zinc</pre> <br />
3.&nbsp; Run generate_h2o.inp once to create temp_h2o.pdb and temp_h2o.mtf. The extra proton atom in the ligand residues, eg. HIS HD1 or CYS S, are removed by editing the generate_h2o.inp; cis proline is also defined here in the generate_h2o.inp (resid is the residue number prior the proline). <br />
<pre>{* any special prosthetic group patches can be applied here *}<br />
{===&gt;}<br />
delete select (name hg and resname cys and resid 61) end<br />
delete select (name hg and resname cys and resid 85) end<br />
delete select (name hd1 and resname his and resid 46) end<br />
delete select (name he2 and resname his and resid 83) end<br />
patch cisp<br />
reference=1=( resid 13 )<br />
end</pre> <br />
4.&nbsp; Edit generate_1.inp to remove the extra proton as did above. <br />
<br />
5.&nbsp; Run generate_20.com, this will run generate_#.inp 20 times, updating each pdb number and this creates cnsPDB/sa_cns_#.pdb <br />
<br />
6.&nbsp; Edit and run re_h2o_cu.inp, the refined pdb is kept in refinedPDB, or <br />
<br />
7.&nbsp; Use subcns to submit cns refinement by using PBS: eg, type "sh subcns". Before run subcns , make a folder " com" contains the following file. Type getpdb to get refined pdb files in refinedPDB after it finished. <br />
<br />
*cns.sc: PBS submission <br />
*cutc_h2o.mtf: mtf file created as descrive above <br />
*topology and parameter files: parallhdg5.3C.pro, parallhdg5.3.pro, topallhdg5.3.pro <br />
*re_h2o_cu.inp: input file for cns refinement<br><br />
<br />
<br> <br />
<br />
=== '''Updated WaterRefCNS script at CABM for use with RDC constraints, metal ions, and dimers''' ===<br />
<br />
In 2011 the WaterRefCNS script was updated by Roberto Tejero.&nbsp; The current implementation uses CNS 1.3 and is capable of performing CNS&nbsp;water refinements including RDC&nbsp;constraints, metal ions constraints and on dimers. <br />
<br />
There are new defaults for the molecular dynamics steps.&nbsp; Again, at CABM&nbsp;we typically run calculations on master3.&nbsp; Here is the list of options and flags, and how the program is launched.&nbsp; Input files can be prepared using the <br />
<br />
[http://www.nmr2.buffalo.edu/nesg.wiki/PdbStat PdbStat] program. <br />
<pre> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ <br />
WaterRefCNS -- a tool to launch structure refinement with water<br />
--Roberto Tejero, 2011<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ <br />
<br />
Usage: /farm/software/WaterRefinement_cns/WaterRefCNS -na NamProt -que [PBS|No] -pro [xpl|cns] <br />
<br />
** Full set of options [not all are needed for a perfect run, use defaults] <br />
<br />
PROTEIN NAME <br />
-di for dimers <br />
-na NamProt Name of the protein (required!) <br />
-seed &lt;int&gt; Seed for random number generator (optional), (def. 31415) <br />
QUEUE SYSTEM AND PROTOCOL<br />
-pr [cns|xpl] Protocol to use, X-POR, CNS def cns <br />
-qu [PBS|NO] Que system to use, one of PBS or NOQUE def NOQUE<br />
PATCHES - SPECIAL RESIDUES <br />
-ci n1,n2,.. CIS pep info (PRO res. num) in comma separated list, i.e 56,89 <br />
-cysn n1,n2,.. CYHN pep (deprotonated SG CYS) res. num in comma separated list, i.e 10,65<br />
-hisd n1,n2,.. HISD pep info (HIS res num) in comma separated list, i.e 12,43 <br />
-hise n1,n2,.. HISE pep info (HIS res num) in comma separated list, i.e 25,67 <br />
CYANA CNS/X-PLOR <br />
~~~~~ ~~~~~~~~~~~ <br />
HIST HISE (HIS with no HD1) <br />
HIS HISD (HIS with no HE2) <br />
HIS+ HIS (HIS with both protons) <br />
<br />
-ss n1-n2,n3-n4 S-S bridge info in comma separated, dash-separated pairs, i.e. 2-24,30-40 <br />
MOLECULAR DYANMICS <br />
-av [center|sum] average for the distance restraints (for CNS, XPLOR) def sum <br />
-cool N number of cycles for cooling stage, default is 100 <br />
-heat N number of cycles in heating stage, default is 200 <br />
-hot N number of cycles in HOT stage, default is 1000 <br />
-par &lt;string&gt; nonbonded params, one of OPLSX|PARAM19|PARMALLH6|CONTACT (def. PARAM19)<br />
-sca N Scale terms (noe, dihe, hbond) by xN times (def. 1.0) <br />
-tsc &lt;real&gt; time step for cooling dynamics (def. 0.003) <br />
-tsh &lt;real&gt; time step for heating dynamics (def. 0.004) <br />
-tsr &lt;real&gt; time step for hot stage dynamics (def. 0.004) <br />
RDC DATA <br />
-DaR1 da,r Da and R for media 1, comma separated values -- overrides automatic detection <br />
-DaR2 da,r Da and R for media 2, comma separated values -- overrides automatic detection <br />
-wrdc1 &lt;real&gt; final weight --ksani1-- for RDC set 1 (def. 0.3) <br />
-wrdc2 &lt;real&gt; final weight --ksani2-- for RDC set 2 (def. 0.3) <br />
SYMMETRY <br />
-sym &lt;string&gt; Activates symmetry, string can be: all | 2:50,52:70 <br />
-wncsi &lt;real&gt; Initial weight for NCS symmetry statement (def 1.0) <br />
-wncsf &lt;real&gt; Final weight for NCS symmetry statement (def 20.0) <br />
HELP - INFO <br />
-he Display this help message <br />
-req Shows the requirements to run this script <br />
-res Displays a short guide to results (file BeSureToReadme) <br />
<br />
Examples: <br />
/farm/software/WaterRefinement_cns/WaterRefCNS -na RTT01 -di -que no -pr cns -ci 22 -hisd 12,42 -cysn 50,89 -wrdc1 1.5 -tsc 0.001 <br />
<br />
<br />
</pre> <br />
Some specific comments: <br />
<br />
1.&nbsp; For calculations using RDC constraints: by default the program uses the Da and R for each individual model in the ensemble.&nbsp; One can override this and enforce a "global" Da an R using the flag: "-DaR1 da,r".&nbsp; <br />
<br />
2.&nbsp; One can also manually adjust the relative weights of the RDCs from different alignments using the "-wrdc1" command.&nbsp; This is often useful when dealing with RDCs from PAG. <br />
<br />
3.&nbsp; Dimers are specified using the flag "-di". <br> <br />
<br />
4.&nbsp; For metal ion constraints, if the constraint file is made using PdbStat be sure to check that the metal ion residue number and charge are correct (see previous section).<br />
<br />
== '''CNS Refinement with RDC constraints, metal ions using FMCGUI at Arrowsmith lab, UofT''' ==<br />
<br />
<font face="Arial, sans-serif"><font size="4">'''Step 1. Input files.'''</font></font> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">In order to perform water refinement using FMCGUI the following files should be prepared/available:</font></font> <br />
<br />
<br> <br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">structure file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">distance constraints file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">dihedral angles constraints file</font></font><br />
<br />
<br> <br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">User can include in the refinement the following additional constrains/data as well:</font></font> <br />
<br />
<br> <br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">hydrogen bond constraints file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data collected in different media; (up to 3 media are allowed)</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Zinc ions</font></font><br />
<br />
<br> <br> <br />
<br />
<br> <br />
<br />
#<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Structure file and constraints file format.</font></font><br><br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">It is expected that the structural, distance constraints, and dihedral angle constraints files are available as output of CYANA2.1 /CYANA3.0 structure calculations. The format of these files correspond to the format of</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">'final.pdb'</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">, 'f</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">inal.upl</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">', and '</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">final.aco'</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">ouput files. At the same time, these files could be loaded in FMCGUI in CNS/XPLOR format as well. It should be noted that structural file should contain key word MODEL that separate lines corresponding to different structural models.</font></font></font></font> <br />
<br />
<u>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Example.</font></font></font></font>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"></font></font></font></u><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">PDB file in CNS format:</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf20.jpeg]] <br />
<br />
&nbsp;<br> <br />
<br />
&nbsp; 2. <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data format</font></font><br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The RDC data can be used in CYANA or PALES formats. One can use RDC data collected in up to 3 media. All data from the same media should be included in the same file. When data are prepared in PALES format than a file should contain data only from one media. When using CYANA3 format, data from different media can be included in one file</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">(See Examples r1,and r2).</font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font><br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example r1.</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data in CYANA3 format.</font></font></font></font> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This example present file with HN and NC rdc's measured in 2 different media.</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf5.jpg]]<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example r2</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">. RDC data in PALES format.</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This example present file with HN, CCA, and NC rdc's measured in 1 media.</font></font></font></font> <br />
<br />
[[Image:Wrf6.jpg]] <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">&nbsp;3.&nbsp; Zinc ligands file format.</font></font><br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The Zinc-ligands file contains list of residues that coordinate each of Zinc ions present in the structure. There could be 3 or 4 Zinc ligands for one Zinc ion. Possible types of residues that coordinate Zinc ion are CYS, HIS, ASP, and GLU.</font></font>&nbsp; <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">(See Example z1)</font></font><br> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example z1</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">. Zinc- ligands file.</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font> <br />
<br />
[[Image:Wrf7.jpg]] <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This file indicate that there are 2 Zinc ions, one Zinc is coordinated by 4 residues 21, 26, 31, and 34 while the other Zinc is coordinated by 3 residues 48, 52, and 62</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This Zinc-file could be created using FMCGUI command [</font></font><font color="#993366"><font face="Times New Roman, serif"><font size="3">'''Structure&gt;Add ZN ligands'''</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">]:</font></font></font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">User have to type IDs of residues that ligate zinc ion into the popped up entry window “ZN ligands” (see Figure). If there are a few zinc ions, information for each ion should be provided in a separate row.</font></font> <br />
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<font face="Arial, sans-serif"><font size="4">'''Step 2. Setting up water refinement calculations using FMCGUI.'''</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">2.1<br />
Open new project [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;new'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]<br />
or load the old one [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;load'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]</span></font></font> <br />
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2.2&nbsp;&nbsp; <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">Select<br />
command</span></span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><br />
[</span></font></font>'''<font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Structure&gt;Water Refinement&gt;calculate</font></font></font>'''<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">]</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The popped up “Water Refinement” window where user is asked to select a number of files with coordinates and constraints (see Figure). The entry fields marked in read (PDB file, Distance constraints, and Dihedral angle constraints) should be specified while the others could be left blank. In addition to coordinates and constraints, user can indicate cisProline residues (if there are any) and to specify singly protonated HIS residues that are double protonated by default.</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">By pressing OK button in “water Refinement” window a new “Parameters” window</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">will pop up. User have to specify</font></font> <br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">name of the water refinement directory</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">flag for distance constraints transformation (could be 1 or 2).&nbsp;</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">If the flag for distance constraints transformation is set to 2 then no change in the upper distance limit is performed. If the flag is set to 1 (default value), then the upper distance limits are modified by classifying each constraint in one of the 4 classes – strong, medium, weak and very weak</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">force constants for RDC constraints (if there are any) on the final stage of the refinement protocol.&nbsp;</span></font></font><br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Pressing OK button starts the setup of water refinement calculations using provided information the specified directory.</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The set up consists of the following operations:</font></font> <br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Splitting structure file in a number of separate files for each model. The atom names are changed from CYANA to CNS nomenclature.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming dihedral angle constraints from CYANA to CNS format. (If constraints are provided in CNS format then no transformation is performed).</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming distance NOE constraints from CYANA format to CNS format. This transformation includes changing atom names from CYANA to CNS nomenclature. If the flag for distance constraints transformation is set to 2 then no change in the upper distance limit is performed. If the flag is set to 1 (default value), then the upper distance limits are modified by classifying each constraint in one of the 4 classes – strong, medium, weak and very weak.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming Hbond constraints (if any) from CYANA format to CNS format</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Generating mtf-file for CNS calculations</font></font><br />
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*"Repairing" each input structure in accord with the mtf-file&nbsp;<br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Generating RDC constraints, a separate file for each media</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Estimating Da and R of alignment tensor for each media. This is performed by fitting RDC data to each model structure using program PALES, with subsequent averaging of the fitting results.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">Generating CNS script "re_h2o*_tmpl.inp" for the refinement calculations<br />
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<br> <font face="Arial, sans-serif"><font size="4">'''Step 3.&nbsp; Water refinement calculations using CNS.'''</font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">In a separate terminal, go to the refinement directory and run the nely generated python script<br />
“</span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">run_refine.py</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">”.</span></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This script will start N jobs in parallel, where N is the number of structural models to refine. The number of structures and the number of processor to use in the calculations can be specified by making modification of the top lines in script “</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">run_refine.py</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">”.<br />
If user want to reset the values for some refinement parameters, for<br />
example the values of Da and R, it could be done by modifying<br />
corresponding lines in the header of the CNS script<br />
</span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">“</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">re_h2o*_tmpl.inp</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”.<br />
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<font face="Arial, sans-serif"><font size="4">'''Step 4. Summarizing results of refinement using FMCGUI.'''</font></font> <br> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">Open<br />
new project [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;new'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]<br />
or load the old one [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;load'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]</span></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">Select<br />
command</span></span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"> </span></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">[</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Structure&gt;Water Refinement&gt;summary'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]&nbsp;&nbsp;&nbsp;&nbsp;</span></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">In the popped up “Water refinement summary” window user will be asked to specify residues used for structure superposition and to select the water refinement directory “WATDIR” were the refinement calculations were made.</font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The refined structural models will be superimpose and combined in one file. The created summary reports values of different energy components and constraint violation statistic for each structural model. A new directory “WATDIR_results” will be created, where WATDIR is the name of selected water refinement directory. The new directory contains final superimposed coordinates, distance and dihedral angle constrains in a format suitable for PDB deposition, summary and constraint violations reports, files</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">“</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">final_cns_summary</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”<br />
and “</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">final_violations</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”<br />
respectively. (See examples S1 and S2)</span></span></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">''<u><span style="font-weight: normal;">Example</span></u>'' <u>S1</u></span><u>.</u> File "final_cns_summary"&nbsp;</font></font></font></font></font></font></font></font></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The file shows the total energy, energy components, and number of constraints violations for each refined structure as well as corresponding average over all structures values.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>[[Image:Wrf12.jpg]]<br></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u><br></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<u>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">ExampleS2.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></u><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">File “</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">final_violations</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">”</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The file contains a list of all constraints violated in at least one structural model. For each constraint the the number of structural models that violate the&nbsp; constraint is shown.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>[[Image:Wrf11.jpg]]<br></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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== <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>'''Files for Download'''</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> ==<br />
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#<u>[[Media:AtomtransC.tbl|atomtransC.tbl]]: atom name translation table</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Agrupa.txt|Agrupa]]: script for concatenating pdb coordinate files into one file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
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#<u>[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Topallhdg5.3.sol|topallhdg5.3.sol]]: CNS solvent topology file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Parallhdg5.3.sol|parallhdg5.3.sol]]: CNS solvent parameter file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, 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#<u>[[Media:Boxtyp20.pdb|boxtyp20.pdb]]: Water box</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Protein.link|protein.link]]: CNS peptide linkage definition file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Generate_water.cns|generate_water.cns]]: CNS protein solvation script</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Print_coorheader.cns|print_coorheader.cns]]: CNS script</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Cns_cabm.tar.gz|cns_cabm.tar.gz]]: CABM CNS refinement protocol</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:WaterRefinement_UB.tar.Z|WaterRefinement_UB.tar.Z]]: CNS water refinement package - SUNY Buffalo</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Cyana2cns.cya|cyana2cns.cya]]: CYANA script to convert coordinates and constrains to XPLOR/CNS format</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, 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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Cu_cPro_cns_example.tar.gz|Cu_cPro_cns_example.tar.gz]]: Example of CNS refinment for protein with Cu and cis-Pro</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, 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#<u>[[Media:Zn.tar|zn.tar]]: Exampleof CNS refinment for protein with zn (from Alex Lemark)</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Er317_2zn_CNSw_example.tar.gz|er317_2zn_CNSw_example.tar.gz]]: Example of CNS refinment for protein with zn (from Theresa Ramelot)</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
<br />
== <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>'''References'''</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> ==<br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>[http://www.ncbi.nlm.nih.gov/pubmed/9757107?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=14 1.&nbsp; Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. and Warren,G.L. (1998) Crystallography &amp; NMR system: a new software suite for macromolecular structure determination. ''Acta Crystallogr. D54'', 905-921.]</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u><br></u> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Structure_Refinement_Using_CNS_Energy_Minimization_With_Explicit_Water&diff=3845
Structure Refinement Using CNS Energy Minimization With Explicit Water
2011-10-05T15:22:23Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
Crystallography &amp; NMR System (CNS) is a program has been designed to provide a flexible multi-level hierachical approach for the most commonly used algorithms in macromolecular structure determination [1]. Functions include heavy atom searching, experimental phasing (including MAD and MIR), density modification, crystallographic refinement with maximum likelihood targets, and NMR structure calculation using NOEs, J-coupling, chemical shift, and dipolar coupling data. <br />
<br />
The latest version is 1.21. Please check the website [http://cns-online.org/ http://cns-online.org] for additional information. <br />
<br />
== '''CNS Refinement Protocol''' ==<br />
<br />
The final structure from CYANA should be refined using CNS energy minimization with explicit water before PDB deposition. <br />
<br />
The common input files are: <br />
<br />
*PDB coordinates <br />
*NOE constraints <br />
*Dihedral angle constraints <br />
*Hydrogen bond constraints<br />
<br />
==== '''Converting input files from CYANA to XPLOR/CNS''' ====<br />
<br />
If the final structure calculation was performed with CYANA 2.1, then the constraint and coordinate files must be first converted to XPLOR/CNS format. <br />
<br />
==== '''Using cyana2cns.cya script''' ====<br />
<br />
This procedure can be used to prepare input files to use with the WaterRefCNS script (see below). <br />
<br />
#Download [[Media:Cyana2cns.cya|cyana2cns.cya]] into directory with the final structure calculation. <br />
#Modify the <tt>read pdb</tt>, <tt>read upl</tt> and <tt>read aco</tt> lines if necessary. <br />
#Start CYANA 2.1 and run <tt>cyana2cns.cya</tt>.<br />
<br />
*All conformers will be stored in a single PDB file <tt>KKK.pdb</tt>. If you plan run water bath refinement manually, use the <tt>p2X</tt> program instead. <br />
*The resulting NOE constraint file <tt>KKK_noe.tbl</tt> will have all lower limits set to 0. If want them to be set according to VdW radii, use the <tt>r2X</tt> program instead. Experience shows that there is no significant effect on geometry and quality scores as reported by PSVS.<br />
<br />
==== '''Using programs p2X and r2X and d2X''' ====<br />
<br />
These programs were written by Alex Lemak at the University of Toronto. They are typically used to prepare input files for manual CNS water bath refinement. <br />
<br />
Go to <tt>~1_projects/targetID/structure/cns/</tt> directory and <tt>conversion_scripts</tt> sub directory, convert required input files to CNS format from CYANA format by using macro <tt>mkfil</tt>:&nbsp; <br />
<pre> p2X cyana2.pdb pref <br />
r2X found-c.upl noeupl.tbl <br />
r2X gr-hbonds.upl hbonds.tbl<br />
cat noeupl.tbl hbonds.tbl &gt; noe.tbl <br />
d2X found-c.aco aco.tbl <br />
</pre> <br />
*<tt>p2X splits conformers from a pdb file generated by CYANA 2.1 into individual pdb files (one file per conformer) and converts atom names to CNS format. The second argument "pref"</tt> specifies a common prefix name for the output pdb files. It should be no more than 4 characters long. <br />
*<tt>r2X</tt> converts distance constraints (noe/hbond) from CYANA 2.1 format to CNS format. The lower limits in the resulting constraint file are set according to VdW radii. <br />
**Distance constraints obtained from CYANA macro <tt>caliba</tt> or <tt>calibration</tt> can be used as input files. <br />
**Distance constraints obtained from CYANA macro <tt>peak calibrate</tt> should have the pseudo-atom correction run before used as input files, run following commands under CYANA: <br> <pre>read upl final.upl<br />
</pre><br />
<br />
distance correct write upl final_corrected.upl This, however, will not add corrections for multiplicity! <br />
<br />
*<tt>d2X</tt>, to run: <tt>d2X cyana2.aco cns.tbl</tt> This will convert angle constraints from CYANA format to CNS format. If you don't have any dihedral angle constrains, create an empty file.<br />
<br />
Both p2X and r2X require a translation table file [[Media:AtomtransC.tbl|atomtransC.tbl]]. <br />
<br />
If have used simplified pseudoatom names (H* instead of Q*) with option pseudo=2 in CYANA 2.1, you may first want to them before running p2X and r2X:<br> <br />
<pre>pseudo=0<br />
read upl noe.upl<br />
read pdb cyana2.pdb<br />
write upl out.upl<br />
write pdb out.pdb all<br />
</pre> <br />
Note that pseudo=0 should be set '''before''' loading PDB and constraint files. <br />
<br />
==== '''Conversion from DYANA''' ====<br />
<br />
Use CYANA macro <tt>MigrateFromDyanaCyana1.cya</tt> to convert of data files from standard CYANA1.x or DYANA nomenclature to the standard IUPAC nomenclature used by CYANA, then do then same thing as above. <br> <br />
<pre> translate dyana # use Cyana 1.x/Dyana nomenclature<br />
read seq demo-dyana.seq # read sequence from Cyana 1.x/Dyana<br />
read upl demo-dyana.upl unknown=warn # read upper distance limits from Cyana 1.x/Dyana<br />
read aco demo-dyana.aco unknown=warn # read angle restraints from Cyana 1.x/Dyana<br />
translate off # return to standard (IUPAC) nomenclature<br />
write demo-cyana.seq # save sequence in standard Cyana format<br />
write upl demo-cyana.upl # save upper distance limits in standard Cyana format<br />
write aco demo-cyana.aco # save angle restraints in standard Cyana format <br />
</pre> <br />
<br> <br />
<br />
=== '''Running CNS using WaterRefCNS script at CABM''' ===<br />
<br />
The WaterRefCNS script, developed at Rutgers University by Roberto Tejero, performs many of the above CNS water refinement protocols in an easy to use script with many user controled options. <br />
<br />
To see a list of options type the command: <br />
<pre> WaterRefCNS<br />
</pre> <br />
You will see a list of options and how the program is used. <br />
<pre> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
WaterRefCN -- a tool to launch structure refinement with water<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
<br />
Usage: /farm/software/WaterRefinement_cns/WaterRefCNS -na NamProt -que [PBS|No] -pro [xpl|cns]<br />
<br />
** Full set of options [not all are needed for a perfect run, use defaults]<br />
<br />
-na NamProt Name of the protein NOTE: this is mandatory, no default value <br />
-qu [PBS|NO] Que system to use, one of PBS or NOQUE def NOQUE<br />
-pr [cns|xpl] Protocol to use, X-POR, CNS def cns <br />
-av [center|sum] average for the distance restraints (for CNS, XPLOR) def sum <br />
-ci n1,n2 CIS pep info (PRO res num) in comma separated list, i.e 56,89 <br />
-ss n1-n2,n3-n4 S-S bridge info in comma separated, dash-separated pairs, i.e. 2-24,30-40 <br />
-he Display this help message<br />
-hisd n1,n2 HISD pep info (HIS res num) in comma separated list, i.e 12,43<br />
-hise n1,n2 HISE pep info (HIS res num) in comma separated list, i.e 25,67<br />
-heat N number of cycles in heating stage, default is 200 <br />
-hot N number of cycles in HOT stage, default is 1000 <br />
-cool N number of cycles for cooling stage, default is 100 <br />
-sc N Scale all terms (noe, dihe, hbond) by xN times def 1 <br />
-par string Choice of nonbonded params, one of OPLSX|PARAM19|PARMALLH6|PROLSQ|CONTACT<br />
default is OPLSX<br />
-seed &lt;int&gt; Seed for random number generator, default 31415 <br />
<br />
Examples:<br />
/farm/software/WaterRefinement_cns/WaterRefCNS -na SR358 -que no -pr cns -ci 22 <br />
<br />
</pre> <br />
Given a protein name KKK, here are the input files required to run WaterRefCNS: <br />
<br />
*&nbsp; pdb coordinates (in a single file) prepared in Xplor format using PdbStat.&nbsp; The name of the file should be:&nbsp; <tt>KKK.pdb</tt> <br />
*&nbsp; an noe.tbl file in CNS format.&nbsp; You can convert CYANA a&nbsp;final.upl file using PdbStat.&nbsp; We typically add 10-15% to the upper limit and make the lower limit vdw.&nbsp; The name of this file should be:&nbsp; <tt>KKK_noe.tbl</tt> <br />
*&nbsp; a dihe.tbl file in CNS&nbsp;format.&nbsp; Again, you can convert a CYANA .aco file using PdbStat.&nbsp; The name of this file should be:&nbsp; <tt>KKK_dihe.tbl</tt> <br />
*&nbsp; an hbond.tbl file in CNS format (optional).&nbsp; The name of this file should be:&nbsp; <tt>KKK_bhond.tbl</tt><br />
<br />
<br> <br />
<br />
In order to run WaterRefCNS on the CABM-NMR cluster: <br />
<br />
#Log into hummer or master3 <br />
#go to the directory with the above 4 files <br />
#type:<br />
<pre> /farm/software/WaterRefinement_cns/WaterRefCNS -na KKK -que PBS</pre> <br />
and that's it.&nbsp; If you have some CIS residue you add <tt>-ci</tt> NUMRES to the options above. If you want to have better vdw violation, you can add <tt>-par PARAM19</tt> to the options above. <br />
<br />
'''Future work:''' <br />
<br />
Currently, the WaterRefCNS script does not support refinement with RDC's.&nbsp; <br />
<br />
<br> <br />
<br />
=== '''WaterRefCNS at SUNY Buffalo''' ===<br />
<br />
The CABM water refinement package was customized to run on computers at UB: <br />
<br />
*Fixed a bug in <tt>WaterRefCNS</tt> so that the assembled refined PDB structure file has a <tt>.pdb</tt> extension. <br />
*PBS queue submission code in <tt>WaterRefCNS</tt> modified for use on U2 cluster <br />
*Added <tt>-hisd</tt> and <tt>-hise</tt> options to use neutral histidine isoprotomers. This required modification of <tt>WaterRefCNS</tt>, <tt>cns_refine_h2o.inp</tt> and <tt>generate_h2o.inp</tt>. <br />
*Fixed a bug in the protein topology file <tt>topallhdg5.3.pro</tt>. The line <tt>atom CD1 type=CR1E charge=0.130 end</tt> in the HISE patch section was removed. <br />
*Fixed a bug that caused the order of conformers to be changed in the output PDB file by replacing the line <tt>$WATREFLIB/Agrupa *.pdb &gt; All_${Name}_cns.pdb</tt> with <tt>$WATREFLIB/Agrupa `ls resa_?.pdb&nbsp;; ls resa_??.pdb` &gt; All_${Name}_cns.pdb</tt> in the <tt>WaterRefCNS</tt> script.<br />
<br />
The modified package is installed in <tt>/nsm/chem/cen2/HTP2/3_src/WaterRefinement_cns</tt> on the local server and in <tt>/san/projects1/szypersk/src/WaterRefinement_cns</tt> on the U2 Linux cluster. Make sure that this directories is included in your path on <tt>spins*</tt> workstations and U2, respectively. <br />
<br />
For reference, the download location is here: [[Media:WaterRefinement_UB.tar.Z|WaterRefinement_UB.tar.Z]] <br />
<br />
The required files are: <br />
<br />
*PDB coordinates (with atom names in XPLOR/CNS format) <br />
*NOE constraints (in XPLOR/CNS format) <br />
*Dihedral angle constraints (in XPLOR/CNS format) <br />
*Hydrogen bond constraints (in XPLOR/CNS format)<br />
<br />
Assuming that your coordinate file is called <tt>KKK.pdb</tt>, the constraint files should be named <tt>KKK_noe.tbl</tt>, <tt>KKK_dihe.tbl</tt> and <tt>KKK_hbond.tbl</tt>. If you don't have dihedral angle and/or hydrogen bond constraints, then create the corresponding empty files. <br />
<br />
You may need to add a line like <tt>nassign=1000</tt> at the top of the constraint files. The value should be equal to or greater than the number of constraints in the file. <br />
<br />
WaterRefCNS assumes "sum of r<sup>-6</sup>" averaging by default. When using "center"-averaged UPLs (e.g. from <tt>caliba</tt>) add <tt>-av center</tt> when starting WaterRefCNS. For information on averaging conventions for calibration in CYANA see [[NOE Calibration Using CYANA|NOE Calibration in CYANA]] <br />
<br />
It is recommended to run it on U2 Linux cluster. Examples: <br />
<br />
#<pre>WaterRefCNS -na KKK -que PBS</pre> <br />
#<pre>WaterRefCNS -na KKK -que PBS -ci 21,49</pre>residues 21 and 49 are cis-Pro <br />
#<pre>WaterRefCNS -na KKK -que PBS -hise 40,82 -hisd 32,65</pre>residues 40 and 82 are ε-protonated neutral His <br> residues 32 and 65 are δ-protonated neutral His <br />
#<pre>WaterRefCNS -na KKK -que PBS -av center </pre>for use with center-averaged calibration<br />
<br />
To run on <tt>spins*</tt> Linux workstations without queue system use <tt>-que NO</tt> option instead of <tt>-que PBS</tt> option. The default is no queue system, so you can omit the <tt>-que</tt> option altogether. <br />
<br />
==== '''Running WaterRefCNS''' ====<br />
<br />
#Convert the coordinate and constraint files as described above. <br />
#Copy the converted files into a CNS working directory on the U2 cluster or a workstation (e.g. <tt>structure/cns/calc1</tt>). <br />
#Run the <tt>WaterRefCNS</tt> with the proper arguments. <br />
#If the calculation was successful, the refined coordinate file will be stored in the <tt>refinedPDB</tt> subdirectory. Check the <tt>BeSureToREADME</tt> for details.<br />
<br />
=== '''Running CNS "manually"''' ===<br />
<br />
Go to <tt>~1_projects/targetID/structure/cns/</tt> directory and <tt>cns_scripts</tt> sub directory, follow the procedures as describe below. <br />
<br />
'''1.&nbsp; Generating MTF topology file from a PDB file. '''<br>&nbsp;&nbsp;&nbsp; modify the header of the input file <tt>generate_mtf.inp</tt> and then run <tt>cns &lt; generate_mtf.inp &gt; mtf.log</tt>. <br />
<br />
*Modify input files path and name base on individual target as follows:<br />
<pre> ...<br />
{in} pdb_file="/san/user/gliu2/u2/cns/cns/convertion_scripts/ufc1_1.pdb";<br />
{in} param_file="/san/user/gliu2/u2/cns/cns/cns_scripts/parallhdg5.3C.pro";<br />
{in} topol_file="/san/user/gliu2/u2/cns/cns/cns_scripts/topallhdg5.3.pro";<br />
{in} plink_file="/san/user/gliu2/u2/cns/cns/cns_scripts/protein.link";<br />
{out} struct_file="ufc1_1.mtf"; <br />
...<br />
</pre> <br />
*Requires these files: <br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file <br />
*[[Media:Protein.link|protein.link]]: CNS peptide linkage definition file <br />
*For <tt>cis-Proline</tt>, define the residue Id of the residue prior to <tt>cis-Proline</tt> by additional lines in <tt>generate_mtf.inp</tt> before the WRITE command<br />
<pre> patch cisp<br />
reference=nil=( resid 90 )<br />
end <br />
</pre> <br />
*If you get an error message on the atom type, you also can try&nbsp;<br />
<pre> patch cipp<br />
reference=nil=( resid 90 )<br />
end </pre> <br />
*For neutral Nε- and Nδ-protonated histidines add these lines in <tt>generate_mtf.inp</tt> before the WRITE command<br />
<pre> patch hise<br />
reference=nil=( resid 40 )<br />
end<br />
patch hisd<br />
reference=nil=( resid 65 )<br />
end </pre> <br />
*For dimer refinement, add a line containing only <tt>TER</tt> between two monomer unit coordinates in the PDB conformer that is defined in <tt>generate_mtf_cis.inp</tt> as input file.<br />
<br />
'''<br>''' <br />
<br />
'''2.&nbsp; Rebuilding hydrogen atom positions for each structure. '''<br>&nbsp;&nbsp;&nbsp; Modify input file <tt>rebuild.inp</tt> and then run <tt>cns &lt; rebuild.inp &gt; rebuild.log</tt> <br />
<br />
*Modify input files path and name base on induvidual target as follows:&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;<br />
<pre> ...<br />
evaluate ($mtf_file="ufc1_1.mtf")<br />
evaluate ($pdbname_in="../convertion_scripts/ufc1")<br />
evaluate ($pdbname_out="ufc1_rb")<br />
evaluate ($number_of_struct= 20 )<br />
...<br />
evaluate ($topol_p_file="/san/user/gliu2/u2/cns/cns/cns_scripts/topallhdg5.3.pro")<br />
evaluate ($param_p_file="/san/user/gliu2/u2/cns/cns/cns_scripts/parallhdg5.3C.pro")<br />
...<br />
</pre> <br />
*Requires these files: <br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file<br />
<br />
'''3. Refining structures with explicit water.''' <br />
<br />
*Running <tt>CNS</tt> on workstation with single processor, modify input file <tt>re_h2oc.inp</tt> and then run <tt>cns &lt; re_h2oc.inp &gt; h2o.log</tt> <br />
*Modify input files path and name base on induvidual target as follows:<br />
<pre> ...<br />
evaluate ($mtf_file="ufc1_1.mtf")<br />
evaluate ($noe_file="../noe.tbl")<br />
evaluate ($dihe_file="../aco.tbl")<br />
evaluate ($hb_file="../hbond.tbl")<br />
evaluate ($pdbname_in="ufc1_rb")<br />
evaluate ($pdbname_out="ufc1_ref")<br />
evaluate ($number_of_struct= 20 )<br />
...</pre> <br />
*Modify the weight of energy contribution part if necessary, e.g. use 2x to 10x of the following scales:<br />
<pre> scale ambi 50<br />
scale dist 50<br />
scale hbond 50<br />
end<br />
restraints dihedral<br />
scale=200<br />
end</pre> <br />
*Requires <br />
**[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
**[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file <br />
**[[Media:Topallhdg5.3.sol|topallhdg5.3.sol]]: CNS solvent topology file <br />
**[[Media:Parallhdg5.3.sol|parallhdg5.3.sol]]: CNS solvent parameter file <br />
**[[Media:Boxtyp20.pdb|boxtyp20.pdb]]: Water box <br />
**[[Media:Generate_water.cns|generate_water.cns]]: CNS protein solvation script <br />
**[[Media:Print_coorheader.cns|print_coorheader.cns]]: CNS script <br />
*Run CNS on workstation with multiple processors. <br> CNS will refine each conformer independently and in parallel, it will be much faster if 20 conformer were refined in parallal at u2.ccr.buffalo.edu.<br />
<br />
&nbsp;&nbsp;&nbsp; 1.&nbsp; Run cns_convertion as descibed above &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp; 2.&nbsp; Go to cns_scripts directory, modify input file <tt>generate_mtf_cis.inp</tt> and then run =cns &lt; generate_mtf_cis.inp = as described above &nbsp;&nbsp;&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp; 3.&nbsp; Run macro <tt>getfil</tt> <br />
<pre> cp ../convertion_scripts/noe.tbl .<br />
cp ../convertion_scripts/hbonds.tbl .<br />
cp ../convertion_scripts/aco.tbl .<br />
cp ufc1_1.mtf com/.</pre> <br />
&nbsp;&nbsp;&nbsp; 4.&nbsp; Modify input file <tt>rebuild.inp</tt> and then run <tt>cns &lt; rebuild.inp</tt> as described above. <br />
<br />
&nbsp;&nbsp;&nbsp; 5.&nbsp; Modify input file <tt>re_h2oc.inp</tt> as described above, remember to set <tt>$number_of_struct = 20</tt>. <br />
<br />
&nbsp;&nbsp;&nbsp; 6.&nbsp; Run macro <tt>subaba</tt> <br />
<br />
<code></code> <br />
<pre> #bash<br />
for i in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20<br />
do<br />
mkdir ref$i<br />
cd ref$i<br />
cp ../com/* .<br />
cp ../jan24/ufc1_rb_$i.pdb ufc1_rb_1.pdb<br />
qsub cns.sc<br />
cd ..<br />
<br />
donedone</pre> <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; where <tt>cns.sc</tt> is a PBS macro used to submit CNS job by using PBS: <br />
<pre> #!/bin/csh<br />
#PBS -m e<br />
#PBS -q short_c<br />
#PBS -l nodes=1:GM:ppn=1<br />
#PBS -l walltime=00:56:00<br />
#PBS -o out1<br />
#PBS -j oe<br />
#PBS -N clean1<br />
cd $PBS_O_WORKDIR<br />
echo "working directory = "$PBS_O_WORKDIR<br />
set NN = `cat $PBS_NODEFILE | wc -l`<br />
echo "NN = "$NN<br />
# Run Job<br />
cns &lt; re_h2oc.inp<br />
echo "ALL Done!"</pre> <br />
&nbsp;&nbsp;&nbsp; 7.&nbsp; Run <tt>getpdb</tt> to collect refined structures. <br />
<pre> #bash<br />
for i in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20<br />
do<br />
cd ref$i<br />
cp ufc1_ref_1.pdb ../pdb/ufc1_ref_$i.pdb<br />
cp ufc1_ref_1.vio ../pdb/ufc1_ref_$i.vio<br />
cd ..<br />
done</pre> <br />
&nbsp;&nbsp;&nbsp; 8. Run script [[Media:Agrupa.txt|Agrupa]] to put all 20 conformers into a single PDB file.<tt></tt> <br />
<pre> Agrupa ufc1_ref*.pdb &gt; ufc1all.pdb</pre> <br />
=== '''Running CNS for Proteins with Metal Ions''' ===<br />
<br />
The CNS refinement for proteins with metal ions can also be performed with the new WaterrefCNS script, using the appropriate metal ion nomenclature in both the pdb coordinates and distance constraints. <br />
<br />
For manual CNS refinement please do the refinement as following: <br />
<br />
1.&nbsp; Set environment for CNS1.1 by run source /farm/users/gliu/alias.cns <br />
<pre> alias cns1 /farm/software/cns/cns_solve_1.1/intel-i686-linux_g77/bin/cns<br />
setenv CNS_TOPPAR /farm/data/gliu/cns1/</pre> <br />
In addition to the topology and parameter files, the metal ion parameter file <tt>ion.top</tt> is required. An example can be found in "/farm/users/gliu/projects/cns_cuttha_cis" with all required input files. 2.&nbsp; Prepared required files as described above (final.tbl, final cns format PDB files and put in xplorPDB dir with name as sa_#.pdb) except the PDB file should include the metal ions with format according to [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/CNS CNS] library <tt>ion.top</tt>. cp sa_1.pdb as&nbsp; template.pdb, input files for creating mtf file Note that alignment is important. eg: <br />
<pre>ATOM 1249 OT2 ALA 83 69.296 13.232 5.744 1.00 0.00<br />
ATOM 1250 ZN+2 ZN2 150 63.086 13.789 -10.407 1.00 0.00 zinc</pre> <br />
3.&nbsp; Run generate_h2o.inp once to create temp_h2o.pdb and temp_h2o.mtf. The extra proton atom in the ligand residues, eg. HIS HD1 or CYS S, are removed by editing the generate_h2o.inp; cis proline is also defined here in the generate_h2o.inp (resid is the residue number prior the proline). <br />
<pre>{* any special prosthetic group patches can be applied here *}<br />
{===&gt;}<br />
delete select (name hg and resname cys and resid 61) end<br />
delete select (name hg and resname cys and resid 85) end<br />
delete select (name hd1 and resname his and resid 46) end<br />
delete select (name he2 and resname his and resid 83) end<br />
patch cisp<br />
reference=1=( resid 13 )<br />
end</pre> <br />
4.&nbsp; Edit generate_1.inp to remove the extra proton as did above. <br />
<br />
5.&nbsp; Run generate_20.com, this will run generate_#.inp 20 times, updating each pdb number and this creates cnsPDB/sa_cns_#.pdb <br />
<br />
6.&nbsp; Edit and run re_h2o_cu.inp, the refined pdb is kept in refinedPDB, or <br />
<br />
7.&nbsp; Use subcns to submit cns refinement by using PBS: eg, type "sh subcns". Before run subcns , make a folder " com" contains the following file. Type getpdb to get refined pdb files in refinedPDB after it finished. <br />
<br />
*cns.sc: PBS submission <br />
*cutc_h2o.mtf: mtf file created as descrive above <br />
*topology and parameter files: parallhdg5.3C.pro, parallhdg5.3.pro, topallhdg5.3.pro <br />
*re_h2o_cu.inp: input file for cns refinement<br><br />
<br />
<br> <br />
<br />
=== '''Updated WaterRefCNS script at CABM for use with RDC constraints, metal ions, and dimers''' ===<br />
<br />
In 2011 the WaterRefCNS script was updated by Roberto Tejero.&nbsp; The current implementation uses CNS 1.3 and is capable of performing CNS&nbsp;water refinements including RDC&nbsp;constraints, metal ions constraints and on dimers. <br />
<br />
There are new defaults for the molecular dynamics steps.&nbsp; Again, at CABM&nbsp;we typically run calculations on master3.&nbsp; Here is the list of options and flags, and how the program is launched.&nbsp; Input files can be prepared using the <br />
<br />
[http://www.nmr2.buffalo.edu/nesg.wiki/PdbStat PdbStat] program. <br />
<pre> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ <br />
WaterRefCNS -- a tool to launch structure refinement with water<br />
--Roberto Tejero, 2011<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ <br />
<br />
Usage: /farm/software/WaterRefinement_cns/WaterRefCNS -na NamProt -que [PBS|No] -pro [xpl|cns] <br />
<br />
** Full set of options [not all are needed for a perfect run, use defaults] <br />
<br />
PROTEIN NAME <br />
-di for dimers <br />
-na NamProt Name of the protein (required!) <br />
-seed &lt;int&gt; Seed for random number generator (optional), (def. 31415) <br />
QUEUE SYSTEM AND PROTOCOL<br />
-pr [cns|xpl] Protocol to use, X-POR, CNS def cns <br />
-qu [PBS|NO] Que system to use, one of PBS or NOQUE def NOQUE<br />
PATCHES - SPECIAL RESIDUES <br />
-ci n1,n2,.. CIS pep info (PRO res. num) in comma separated list, i.e 56,89 <br />
-cysn n1,n2,.. CYHN pep (deprotonated SG CYS) res. num in comma separated list, i.e 10,65<br />
-hisd n1,n2,.. HISD pep info (HIS res num) in comma separated list, i.e 12,43 <br />
-hise n1,n2,.. HISE pep info (HIS res num) in comma separated list, i.e 25,67 <br />
CYANA CNS/X-PLOR <br />
~~~~~ ~~~~~~~~~~~ <br />
HIST HISE (HIS with no HD1) <br />
HIS HISD (HIS with no HE2) <br />
HIS+ HIS (HIS with both protons) <br />
<br />
-ss n1-n2,n3-n4 S-S bridge info in comma separated, dash-separated pairs, i.e. 2-24,30-40 <br />
MOLECULAR DYANMICS <br />
-av [center|sum] average for the distance restraints (for CNS, XPLOR) def sum <br />
-cool N number of cycles for cooling stage, default is 100 <br />
-heat N number of cycles in heating stage, default is 200 <br />
-hot N number of cycles in HOT stage, default is 1000 <br />
-par &lt;string&gt; nonbonded params, one of OPLSX|PARAM19|PARMALLH6|CONTACT (def. PARAM19)<br />
-sca N Scale terms (noe, dihe, hbond) by xN times (def. 1.0) <br />
-tsc &lt;real&gt; time step for cooling dynamics (def. 0.003) <br />
-tsh &lt;real&gt; time step for heating dynamics (def. 0.004) <br />
-tsr &lt;real&gt; time step for hot stage dynamics (def. 0.004) <br />
RDC DATA <br />
-DaR1 da,r Da and R for media 1, comma separated values -- overrides automatic detection <br />
-DaR2 da,r Da and R for media 2, comma separated values -- overrides automatic detection <br />
-wrdc1 &lt;real&gt; final weight --ksani1-- for RDC set 1 (def. 0.3) <br />
-wrdc2 &lt;real&gt; final weight --ksani2-- for RDC set 2 (def. 0.3) <br />
SYMMETRY <br />
-sym &lt;string&gt; Activates symmetry, string can be: all | 2:50,52:70 <br />
-wncsi &lt;real&gt; Initial weight for NCS symmetry statement (def 1.0) <br />
-wncsf &lt;real&gt; Final weight for NCS symmetry statement (def 20.0) <br />
HELP - INFO <br />
-he Display this help message <br />
-req Shows the requirements to run this script <br />
-res Displays a short guide to results (file BeSureToReadme) <br />
<br />
Examples: <br />
/farm/software/WaterRefinement_cns/WaterRefCNS -na RTT01 -di -que no -pr cns -ci 22 -hisd 12,42 -cysn 50,89 -wrdc1 1.5 -tsc 0.001 <br />
<br />
<br />
</pre> <br />
Some specific comments: <br />
<br />
1.&nbsp; For calculations using RDC constraints: by default the program uses the Da and R for each individual model in the ensemble.&nbsp; One can override this and enforce a "global" Da an R using the flag: <br />
<br />
"-DaR1 da,r".&nbsp; <br />
<br />
2.&nbsp; One can also manually adjust the relative weights of the RDCs from different alignments using the "-wrdc1" command.&nbsp; This is often useful when dealing with RDCs from PAG. <br />
<br />
3.&nbsp; Dimers are specified using the flag "-di". <br> <br />
<br />
== '''CNS Refinement with RDC constraints, metal ions using FMCGUI at Arrowsmith lab, UofT''' ==<br />
<br />
<font face="Arial, sans-serif"><font size="4">'''Step 1. Input files.'''</font></font> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">In order to perform water refinement using FMCGUI the following files should be prepared/available:</font></font> <br />
<br />
<br> <br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">structure file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">distance constraints file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">dihedral angles constraints file</font></font><br />
<br />
<br> <br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">User can include in the refinement the following additional constrains/data as well:</font></font> <br />
<br />
<br> <br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">hydrogen bond constraints file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data collected in different media; (up to 3 media are allowed)</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Zinc ions</font></font><br />
<br />
<br> <br> <br />
<br />
<br> <br />
<br />
#<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Structure file and constraints file format.</font></font><br><br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">It is expected that the structural, distance constraints, and dihedral angle constraints files are available as output of CYANA2.1 /CYANA3.0 structure calculations. The format of these files correspond to the format of</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">'final.pdb'</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">, 'f</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">inal.upl</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">', and '</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">final.aco'</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">ouput files. At the same time, these files could be loaded in FMCGUI in CNS/XPLOR format as well. It should be noted that structural file should contain key word MODEL that separate lines corresponding to different structural models.</font></font></font></font> <br />
<br />
<u>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Example.</font></font></font></font>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"></font></font></font></u><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">PDB file in CNS format:</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf20.jpeg]] <br />
<br />
&nbsp;<br> <br />
<br />
&nbsp; 2. <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data format</font></font><br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The RDC data can be used in CYANA or PALES formats. One can use RDC data collected in up to 3 media. All data from the same media should be included in the same file. When data are prepared in PALES format than a file should contain data only from one media. When using CYANA3 format, data from different media can be included in one file</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">(See Examples r1,and r2).</font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font><br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example r1.</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data in CYANA3 format.</font></font></font></font> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This example present file with HN and NC rdc's measured in 2 different media.</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf5.jpg]]<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example r2</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">. RDC data in PALES format.</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This example present file with HN, CCA, and NC rdc's measured in 1 media.</font></font></font></font> <br />
<br />
[[Image:Wrf6.jpg]] <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">&nbsp;3.&nbsp; Zinc ligands file format.</font></font><br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The Zinc-ligands file contains list of residues that coordinate each of Zinc ions present in the structure. There could be 3 or 4 Zinc ligands for one Zinc ion. Possible types of residues that coordinate Zinc ion are CYS, HIS, ASP, and GLU.</font></font>&nbsp; <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">(See Example z1)</font></font><br> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example z1</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">. Zinc- ligands file.</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font> <br />
<br />
[[Image:Wrf7.jpg]] <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This file indicate that there are 2 Zinc ions, one Zinc is coordinated by 4 residues 21, 26, 31, and 34 while the other Zinc is coordinated by 3 residues 48, 52, and 62</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This Zinc-file could be created using FMCGUI command [</font></font><font color="#993366"><font face="Times New Roman, serif"><font size="3">'''Structure&gt;Add ZN ligands'''</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">]:</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf8.jpg]]<br> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">User have to type IDs of residues that ligate zinc ion into the popped up entry window “ZN ligands” (see Figure). If there are a few zinc ions, information for each ion should be provided in a separate row.</font></font> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="4">'''Step 2. Setting up water refinement calculations using FMCGUI.'''</font></font> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">2.1<br />
Open new project [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;new'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]<br />
or load the old one [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;load'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]</span></font></font> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
2.2&nbsp;&nbsp; <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">Select<br />
command</span></span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><br />
[</span></font></font>'''<font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Structure&gt;Water Refinement&gt;calculate</font></font></font>'''<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">]</font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">[[Image:Wrf14.jpg]]</font></font> <br />
<br />
<br> <br> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The popped up “Water Refinement” window where user is asked to select a number of files with coordinates and constraints (see Figure). The entry fields marked in read (PDB file, Distance constraints, and Dihedral angle constraints) should be specified while the others could be left blank. In addition to coordinates and constraints, user can indicate cisProline residues (if there are any) and to specify singly protonated HIS residues that are double protonated by default.</font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf18.jpeg]]<br> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">By pressing OK button in “water Refinement” window a new “Parameters” window</font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">will pop up. User have to specify</font></font> <br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">name of the water refinement directory</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">flag for distance constraints transformation (could be 1 or 2).&nbsp;</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">If the flag for distance constraints transformation is set to 2 then no change in the upper distance limit is performed. If the flag is set to 1 (default value), then the upper distance limits are modified by classifying each constraint in one of the 4 classes – strong, medium, weak and very weak</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">force constants for RDC constraints (if there are any) on the final stage of the refinement protocol.&nbsp;</span></font></font><br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<br> [[Image:Wrf10.jpg]]<br> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Pressing OK button starts the setup of water refinement calculations using provided information the specified directory.</font></font> <br />
<br />
<br> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The set up consists of the following operations:</font></font> <br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Splitting structure file in a number of separate files for each model. The atom names are changed from CYANA to CNS nomenclature.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming dihedral angle constraints from CYANA to CNS format. (If constraints are provided in CNS format then no transformation is performed).</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming distance NOE constraints from CYANA format to CNS format. This transformation includes changing atom names from CYANA to CNS nomenclature. If the flag for distance constraints transformation is set to 2 then no change in the upper distance limit is performed. If the flag is set to 1 (default value), then the upper distance limits are modified by classifying each constraint in one of the 4 classes – strong, medium, weak and very weak.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming Hbond constraints (if any) from CYANA format to CNS format</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Generating mtf-file for CNS calculations</font></font><br />
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*"Repairing" each input structure in accord with the mtf-file&nbsp;<br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Generating RDC constraints, a separate file for each media</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Estimating Da and R of alignment tensor for each media. This is performed by fitting RDC data to each model structure using program PALES, with subsequent averaging of the fitting results.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">Generating CNS script "re_h2o*_tmpl.inp" for the refinement calculations<br />
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<br> <font face="Arial, sans-serif"><font size="4">'''Step 3.&nbsp; Water refinement calculations using CNS.'''</font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">In a separate terminal, go to the refinement directory and run the nely generated python script<br />
“</span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">run_refine.py</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">”.</span></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This script will start N jobs in parallel, where N is the number of structural models to refine. The number of structures and the number of processor to use in the calculations can be specified by making modification of the top lines in script “</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">run_refine.py</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">”.<br />
If user want to reset the values for some refinement parameters, for<br />
example the values of Da and R, it could be done by modifying<br />
corresponding lines in the header of the CNS script<br />
</span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">“</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">re_h2o*_tmpl.inp</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”.<br />
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<font face="Arial, sans-serif"><font size="4">'''Step 4. Summarizing results of refinement using FMCGUI.'''</font></font> <br> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">Open<br />
new project [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;new'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]<br />
or load the old one [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;load'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]</span></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">Select<br />
command</span></span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"> </span></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">[</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Structure&gt;Water Refinement&gt;summary'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]&nbsp;&nbsp;&nbsp;&nbsp;</span></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br />
</font></font></font></font></span></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">In the popped up “Water refinement summary” window user will be asked to specify residues used for structure superposition and to select the water refinement directory “WATDIR” were the refinement calculations were made.</font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font>[[Image:Wrf19.jpg]] <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The refined structural models will be superimpose and combined in one file. The created summary reports values of different energy components and constraint violation statistic for each structural model. A new directory “WATDIR_results” will be created, where WATDIR is the name of selected water refinement directory. The new directory contains final superimposed coordinates, distance and dihedral angle constrains in a format suitable for PDB deposition, summary and constraint violations reports, files</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">“</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">final_cns_summary</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”<br />
and “</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">final_violations</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”<br />
respectively. (See examples S1 and S2)</span></span></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">''<u><span style="font-weight: normal;">Example</span></u>'' <u>S1</u></span><u>.</u> File "final_cns_summary"&nbsp;</font></font></font></font></font></font></font></font></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The file shows the total energy, energy components, and number of constraints violations for each refined structure as well as corresponding average over all structures values.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<u>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">ExampleS2.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></u><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">File “</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">final_violations</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">”</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The file contains a list of all constraints violated in at least one structural model. For each constraint the the number of structural models that violate the&nbsp; constraint is shown.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>[[Image:Wrf11.jpg]]<br></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u><br></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u><br></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u><br></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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== <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>'''Files for Download'''</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> ==<br />
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#<u>[[Media:AtomtransC.tbl|atomtransC.tbl]]: atom name translation table</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Agrupa.txt|Agrupa]]: script for concatenating pdb coordinate files into one file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
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#<u>[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
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#<u>[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
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#<u>[[Media:Topallhdg5.3.sol|topallhdg5.3.sol]]: CNS solvent topology file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 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New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
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#<u>[[Media:Parallhdg5.3.sol|parallhdg5.3.sol]]: CNS solvent parameter file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Boxtyp20.pdb|boxtyp20.pdb]]: Water box</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Protein.link|protein.link]]: CNS peptide linkage definition file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Generate_water.cns|generate_water.cns]]: CNS protein solvation script</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Print_coorheader.cns|print_coorheader.cns]]: CNS script</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Cns_cabm.tar.gz|cns_cabm.tar.gz]]: CABM CNS refinement protocol</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:WaterRefinement_UB.tar.Z|WaterRefinement_UB.tar.Z]]: CNS water refinement package - SUNY Buffalo</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Cyana2cns.cya|cyana2cns.cya]]: CYANA script to convert coordinates and constrains to XPLOR/CNS format</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Cu_cPro_cns_example.tar.gz|Cu_cPro_cns_example.tar.gz]]: Example of CNS refinment for protein with Cu and cis-Pro</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Zn.tar|zn.tar]]: Exampleof CNS refinment for protein with zn (from Alex Lemark)</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Er317_2zn_CNSw_example.tar.gz|er317_2zn_CNSw_example.tar.gz]]: Example of CNS refinment for protein with zn (from Theresa Ramelot)</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
<br />
== <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>'''References'''</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> ==<br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>[http://www.ncbi.nlm.nih.gov/pubmed/9757107?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=14 1.&nbsp; Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. and Warren,G.L. (1998) Crystallography &amp; NMR system: a new software suite for macromolecular structure determination. ''Acta Crystallogr. D54'', 905-921.]</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u><br></u> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Structure_Refinement_Using_CNS_Energy_Minimization_With_Explicit_Water&diff=3844
Structure Refinement Using CNS Energy Minimization With Explicit Water
2011-10-05T15:19:11Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
Crystallography &amp; NMR System (CNS) is a program has been designed to provide a flexible multi-level hierachical approach for the most commonly used algorithms in macromolecular structure determination [1]. Functions include heavy atom searching, experimental phasing (including MAD and MIR), density modification, crystallographic refinement with maximum likelihood targets, and NMR structure calculation using NOEs, J-coupling, chemical shift, and dipolar coupling data. <br />
<br />
The latest version is 1.21. Please check the website [http://cns-online.org/ http://cns-online.org] for additional information. <br />
<br />
== '''CNS Refinement Protocol''' ==<br />
<br />
The final structure from CYANA should be refined using CNS energy minimization with explicit water before PDB deposition. <br />
<br />
The common input files are: <br />
<br />
*PDB coordinates <br />
*NOE constraints <br />
*Dihedral angle constraints <br />
*Hydrogen bond constraints<br />
<br />
==== '''Converting input files from CYANA to XPLOR/CNS''' ====<br />
<br />
If the final structure calculation was performed with CYANA 2.1, then the constraint and coordinate files must be first converted to XPLOR/CNS format. <br />
<br />
==== '''Using cyana2cns.cya script''' ====<br />
<br />
This procedure can be used to prepare input files to use with the WaterRefCNS script (see below). <br />
<br />
#Download [[Media:Cyana2cns.cya|cyana2cns.cya]] into directory with the final structure calculation. <br />
#Modify the <tt>read pdb</tt>, <tt>read upl</tt> and <tt>read aco</tt> lines if necessary. <br />
#Start CYANA 2.1 and run <tt>cyana2cns.cya</tt>.<br />
<br />
*All conformers will be stored in a single PDB file <tt>KKK.pdb</tt>. If you plan run water bath refinement manually, use the <tt>p2X</tt> program instead. <br />
*The resulting NOE constraint file <tt>KKK_noe.tbl</tt> will have all lower limits set to 0. If want them to be set according to VdW radii, use the <tt>r2X</tt> program instead. Experience shows that there is no significant effect on geometry and quality scores as reported by PSVS.<br />
<br />
==== '''Using programs p2X and r2X and d2X''' ====<br />
<br />
These programs were written by Alex Lemak at the University of Toronto. They are typically used to prepare input files for manual CNS water bath refinement. <br />
<br />
Go to <tt>~1_projects/targetID/structure/cns/</tt> directory and <tt>conversion_scripts</tt> sub directory, convert required input files to CNS format from CYANA format by using macro <tt>mkfil</tt>:&nbsp; <br />
<pre> p2X cyana2.pdb pref <br />
r2X found-c.upl noeupl.tbl <br />
r2X gr-hbonds.upl hbonds.tbl<br />
cat noeupl.tbl hbonds.tbl &gt; noe.tbl <br />
d2X found-c.aco aco.tbl <br />
</pre> <br />
*<tt>p2X splits conformers from a pdb file generated by CYANA 2.1 into individual pdb files (one file per conformer) and converts atom names to CNS format. The second argument "pref"</tt> specifies a common prefix name for the output pdb files. It should be no more than 4 characters long. <br />
*<tt>r2X</tt> converts distance constraints (noe/hbond) from CYANA 2.1 format to CNS format. The lower limits in the resulting constraint file are set according to VdW radii. <br />
**Distance constraints obtained from CYANA macro <tt>caliba</tt> or <tt>calibration</tt> can be used as input files. <br />
**Distance constraints obtained from CYANA macro <tt>peak calibrate</tt> should have the pseudo-atom correction run before used as input files, run following commands under CYANA: <br> <pre>read upl final.upl<br />
</pre><br />
<br />
distance correct write upl final_corrected.upl This, however, will not add corrections for multiplicity! <br />
<br />
*<tt>d2X</tt>, to run: <tt>d2X cyana2.aco cns.tbl</tt> This will convert angle constraints from CYANA format to CNS format. If you don't have any dihedral angle constrains, create an empty file.<br />
<br />
Both p2X and r2X require a translation table file [[Media:AtomtransC.tbl|atomtransC.tbl]]. <br />
<br />
If have used simplified pseudoatom names (H* instead of Q*) with option pseudo=2 in CYANA 2.1, you may first want to them before running p2X and r2X:<br> <br />
<pre>pseudo=0<br />
read upl noe.upl<br />
read pdb cyana2.pdb<br />
write upl out.upl<br />
write pdb out.pdb all<br />
</pre> <br />
Note that pseudo=0 should be set '''before''' loading PDB and constraint files. <br />
<br />
==== '''Conversion from DYANA''' ====<br />
<br />
Use CYANA macro <tt>MigrateFromDyanaCyana1.cya</tt> to convert of data files from standard CYANA1.x or DYANA nomenclature to the standard IUPAC nomenclature used by CYANA, then do then same thing as above. <br> <br />
<pre> translate dyana # use Cyana 1.x/Dyana nomenclature<br />
read seq demo-dyana.seq # read sequence from Cyana 1.x/Dyana<br />
read upl demo-dyana.upl unknown=warn # read upper distance limits from Cyana 1.x/Dyana<br />
read aco demo-dyana.aco unknown=warn # read angle restraints from Cyana 1.x/Dyana<br />
translate off # return to standard (IUPAC) nomenclature<br />
write demo-cyana.seq # save sequence in standard Cyana format<br />
write upl demo-cyana.upl # save upper distance limits in standard Cyana format<br />
write aco demo-cyana.aco # save angle restraints in standard Cyana format <br />
</pre> <br />
<br> <br />
<br />
=== '''Running CNS using WaterRefCNS script at CABM''' ===<br />
<br />
The WaterRefCNS script, developed at Rutgers University by Roberto Tejero, performs many of the above CNS water refinement protocols in an easy to use script with many user controled options. <br />
<br />
To see a list of options type the command: <br />
<pre> WaterRefCNS<br />
</pre> <br />
You will see a list of options and how the program is used. <br />
<pre> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
WaterRefCN -- a tool to launch structure refinement with water<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
<br />
Usage: /farm/software/WaterRefinement_cns/WaterRefCNS -na NamProt -que [PBS|No] -pro [xpl|cns]<br />
<br />
** Full set of options [not all are needed for a perfect run, use defaults]<br />
<br />
-na NamProt Name of the protein NOTE: this is mandatory, no default value <br />
-qu [PBS|NO] Que system to use, one of PBS or NOQUE def NOQUE<br />
-pr [cns|xpl] Protocol to use, X-POR, CNS def cns <br />
-av [center|sum] average for the distance restraints (for CNS, XPLOR) def sum <br />
-ci n1,n2 CIS pep info (PRO res num) in comma separated list, i.e 56,89 <br />
-ss n1-n2,n3-n4 S-S bridge info in comma separated, dash-separated pairs, i.e. 2-24,30-40 <br />
-he Display this help message<br />
-hisd n1,n2 HISD pep info (HIS res num) in comma separated list, i.e 12,43<br />
-hise n1,n2 HISE pep info (HIS res num) in comma separated list, i.e 25,67<br />
-heat N number of cycles in heating stage, default is 200 <br />
-hot N number of cycles in HOT stage, default is 1000 <br />
-cool N number of cycles for cooling stage, default is 100 <br />
-sc N Scale all terms (noe, dihe, hbond) by xN times def 1 <br />
-par string Choice of nonbonded params, one of OPLSX|PARAM19|PARMALLH6|PROLSQ|CONTACT<br />
default is OPLSX<br />
-seed &lt;int&gt; Seed for random number generator, default 31415 <br />
<br />
Examples:<br />
/farm/software/WaterRefinement_cns/WaterRefCNS -na SR358 -que no -pr cns -ci 22 <br />
<br />
</pre> <br />
Given a protein name KKK, here are the input files required to run WaterRefCNS: <br />
<br />
*&nbsp; pdb coordinates (in a single file) prepared in Xplor format using PdbStat.&nbsp; The name of the file should be:&nbsp; <tt>KKK.pdb</tt> <br />
*&nbsp; an noe.tbl file in CNS format.&nbsp; You can convert CYANA a&nbsp;final.upl file using PdbStat.&nbsp; We typically add 10-15% to the upper limit and make the lower limit vdw.&nbsp; The name of this file should be:&nbsp; <tt>KKK_noe.tbl</tt> <br />
*&nbsp; a dihe.tbl file in CNS&nbsp;format.&nbsp; Again, you can convert a CYANA .aco file using PdbStat.&nbsp; The name of this file should be:&nbsp; <tt>KKK_dihe.tbl</tt> <br />
*&nbsp; an hbond.tbl file in CNS format (optional).&nbsp; The name of this file should be:&nbsp; <tt>KKK_bhond.tbl</tt><br />
<br />
<br> <br />
<br />
In order to run WaterRefCNS on the CABM-NMR cluster: <br />
<br />
#Log into hummer or master3 <br />
#go to the directory with the above 4 files <br />
#type:<br />
<pre> /farm/software/WaterRefinement_cns/WaterRefCNS -na KKK -que PBS</pre> <br />
and that's it.&nbsp; If you have some CIS residue you add <tt>-ci</tt> NUMRES to the options above. If you want to have better vdw violation, you can add <tt>-par PARAM19</tt> to the options above. <br />
<br />
'''Future work:''' <br />
<br />
Currently, the WaterRefCNS script does not support refinement with RDC's.&nbsp; <br />
<br />
<br> <br />
<br />
=== '''WaterRefCNS at SUNY Buffalo''' ===<br />
<br />
The CABM water refinement package was customized to run on computers at UB: <br />
<br />
*Fixed a bug in <tt>WaterRefCNS</tt> so that the assembled refined PDB structure file has a <tt>.pdb</tt> extension. <br />
*PBS queue submission code in <tt>WaterRefCNS</tt> modified for use on U2 cluster <br />
*Added <tt>-hisd</tt> and <tt>-hise</tt> options to use neutral histidine isoprotomers. This required modification of <tt>WaterRefCNS</tt>, <tt>cns_refine_h2o.inp</tt> and <tt>generate_h2o.inp</tt>. <br />
*Fixed a bug in the protein topology file <tt>topallhdg5.3.pro</tt>. The line <tt>atom CD1 type=CR1E charge=0.130 end</tt> in the HISE patch section was removed. <br />
*Fixed a bug that caused the order of conformers to be changed in the output PDB file by replacing the line <tt>$WATREFLIB/Agrupa *.pdb &gt; All_${Name}_cns.pdb</tt> with <tt>$WATREFLIB/Agrupa `ls resa_?.pdb&nbsp;; ls resa_??.pdb` &gt; All_${Name}_cns.pdb</tt> in the <tt>WaterRefCNS</tt> script.<br />
<br />
The modified package is installed in <tt>/nsm/chem/cen2/HTP2/3_src/WaterRefinement_cns</tt> on the local server and in <tt>/san/projects1/szypersk/src/WaterRefinement_cns</tt> on the U2 Linux cluster. Make sure that this directories is included in your path on <tt>spins*</tt> workstations and U2, respectively. <br />
<br />
For reference, the download location is here: [[Media:WaterRefinement_UB.tar.Z|WaterRefinement_UB.tar.Z]] <br />
<br />
The required files are: <br />
<br />
*PDB coordinates (with atom names in XPLOR/CNS format) <br />
*NOE constraints (in XPLOR/CNS format) <br />
*Dihedral angle constraints (in XPLOR/CNS format) <br />
*Hydrogen bond constraints (in XPLOR/CNS format)<br />
<br />
Assuming that your coordinate file is called <tt>KKK.pdb</tt>, the constraint files should be named <tt>KKK_noe.tbl</tt>, <tt>KKK_dihe.tbl</tt> and <tt>KKK_hbond.tbl</tt>. If you don't have dihedral angle and/or hydrogen bond constraints, then create the corresponding empty files. <br />
<br />
You may need to add a line like <tt>nassign=1000</tt> at the top of the constraint files. The value should be equal to or greater than the number of constraints in the file. <br />
<br />
WaterRefCNS assumes "sum of r<sup>-6</sup>" averaging by default. When using "center"-averaged UPLs (e.g. from <tt>caliba</tt>) add <tt>-av center</tt> when starting WaterRefCNS. For information on averaging conventions for calibration in CYANA see [[NOE Calibration Using CYANA|NOE Calibration in CYANA]] <br />
<br />
It is recommended to run it on U2 Linux cluster. Examples: <br />
<br />
#<pre>WaterRefCNS -na KKK -que PBS</pre> <br />
#<pre>WaterRefCNS -na KKK -que PBS -ci 21,49</pre>residues 21 and 49 are cis-Pro <br />
#<pre>WaterRefCNS -na KKK -que PBS -hise 40,82 -hisd 32,65</pre>residues 40 and 82 are ε-protonated neutral His <br> residues 32 and 65 are δ-protonated neutral His <br />
#<pre>WaterRefCNS -na KKK -que PBS -av center </pre>for use with center-averaged calibration<br />
<br />
To run on <tt>spins*</tt> Linux workstations without queue system use <tt>-que NO</tt> option instead of <tt>-que PBS</tt> option. The default is no queue system, so you can omit the <tt>-que</tt> option altogether. <br />
<br />
==== '''Running WaterRefCNS''' ====<br />
<br />
#Convert the coordinate and constraint files as described above. <br />
#Copy the converted files into a CNS working directory on the U2 cluster or a workstation (e.g. <tt>structure/cns/calc1</tt>). <br />
#Run the <tt>WaterRefCNS</tt> with the proper arguments. <br />
#If the calculation was successful, the refined coordinate file will be stored in the <tt>refinedPDB</tt> subdirectory. Check the <tt>BeSureToREADME</tt> for details.<br />
<br />
=== '''Running CNS "manually"''' ===<br />
<br />
Go to <tt>~1_projects/targetID/structure/cns/</tt> directory and <tt>cns_scripts</tt> sub directory, follow the procedures as describe below. <br />
<br />
'''1.&nbsp; Generating MTF topology file from a PDB file. '''<br>&nbsp;&nbsp;&nbsp; modify the header of the input file <tt>generate_mtf.inp</tt> and then run <tt>cns &lt; generate_mtf.inp &gt; mtf.log</tt>. <br />
<br />
*Modify input files path and name base on individual target as follows:<br />
<pre> ...<br />
{in} pdb_file="/san/user/gliu2/u2/cns/cns/convertion_scripts/ufc1_1.pdb";<br />
{in} param_file="/san/user/gliu2/u2/cns/cns/cns_scripts/parallhdg5.3C.pro";<br />
{in} topol_file="/san/user/gliu2/u2/cns/cns/cns_scripts/topallhdg5.3.pro";<br />
{in} plink_file="/san/user/gliu2/u2/cns/cns/cns_scripts/protein.link";<br />
{out} struct_file="ufc1_1.mtf"; <br />
...<br />
</pre> <br />
*Requires these files: <br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file <br />
*[[Media:Protein.link|protein.link]]: CNS peptide linkage definition file <br />
*For <tt>cis-Proline</tt>, define the residue Id of the residue prior to <tt>cis-Proline</tt> by additional lines in <tt>generate_mtf.inp</tt> before the WRITE command<br />
<pre> patch cisp<br />
reference=nil=( resid 90 )<br />
end <br />
</pre> <br />
*If you get an error message on the atom type, you also can try&nbsp;<br />
<pre> patch cipp<br />
reference=nil=( resid 90 )<br />
end </pre> <br />
*For neutral Nε- and Nδ-protonated histidines add these lines in <tt>generate_mtf.inp</tt> before the WRITE command<br />
<pre> patch hise<br />
reference=nil=( resid 40 )<br />
end<br />
patch hisd<br />
reference=nil=( resid 65 )<br />
end </pre> <br />
*For dimer refinement, add a line containing only <tt>TER</tt> between two monomer unit coordinates in the PDB conformer that is defined in <tt>generate_mtf_cis.inp</tt> as input file.<br />
<br />
'''<br>''' <br />
<br />
'''2.&nbsp; Rebuilding hydrogen atom positions for each structure. '''<br>&nbsp;&nbsp;&nbsp; Modify input file <tt>rebuild.inp</tt> and then run <tt>cns &lt; rebuild.inp &gt; rebuild.log</tt> <br />
<br />
*Modify input files path and name base on induvidual target as follows:&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;<br />
<pre> ...<br />
evaluate ($mtf_file="ufc1_1.mtf")<br />
evaluate ($pdbname_in="../convertion_scripts/ufc1")<br />
evaluate ($pdbname_out="ufc1_rb")<br />
evaluate ($number_of_struct= 20 )<br />
...<br />
evaluate ($topol_p_file="/san/user/gliu2/u2/cns/cns/cns_scripts/topallhdg5.3.pro")<br />
evaluate ($param_p_file="/san/user/gliu2/u2/cns/cns/cns_scripts/parallhdg5.3C.pro")<br />
...<br />
</pre> <br />
*Requires these files: <br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file<br />
<br />
'''3. Refining structures with explicit water.''' <br />
<br />
*Running <tt>CNS</tt> on workstation with single processor, modify input file <tt>re_h2oc.inp</tt> and then run <tt>cns &lt; re_h2oc.inp &gt; h2o.log</tt> <br />
*Modify input files path and name base on induvidual target as follows:<br />
<pre> ...<br />
evaluate ($mtf_file="ufc1_1.mtf")<br />
evaluate ($noe_file="../noe.tbl")<br />
evaluate ($dihe_file="../aco.tbl")<br />
evaluate ($hb_file="../hbond.tbl")<br />
evaluate ($pdbname_in="ufc1_rb")<br />
evaluate ($pdbname_out="ufc1_ref")<br />
evaluate ($number_of_struct= 20 )<br />
...</pre> <br />
*Modify the weight of energy contribution part if necessary, e.g. use 2x to 10x of the following scales:<br />
<pre> scale ambi 50<br />
scale dist 50<br />
scale hbond 50<br />
end<br />
restraints dihedral<br />
scale=200<br />
end</pre> <br />
*Requires <br />
**[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
**[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file <br />
**[[Media:Topallhdg5.3.sol|topallhdg5.3.sol]]: CNS solvent topology file <br />
**[[Media:Parallhdg5.3.sol|parallhdg5.3.sol]]: CNS solvent parameter file <br />
**[[Media:Boxtyp20.pdb|boxtyp20.pdb]]: Water box <br />
**[[Media:Generate_water.cns|generate_water.cns]]: CNS protein solvation script <br />
**[[Media:Print_coorheader.cns|print_coorheader.cns]]: CNS script <br />
*Run CNS on workstation with multiple processors. <br> CNS will refine each conformer independently and in parallel, it will be much faster if 20 conformer were refined in parallal at u2.ccr.buffalo.edu.<br />
<br />
&nbsp;&nbsp;&nbsp; 1.&nbsp; Run cns_convertion as descibed above &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp; 2.&nbsp; Go to cns_scripts directory, modify input file <tt>generate_mtf_cis.inp</tt> and then run =cns &lt; generate_mtf_cis.inp = as described above &nbsp;&nbsp;&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp; 3.&nbsp; Run macro <tt>getfil</tt> <br />
<pre> cp ../convertion_scripts/noe.tbl .<br />
cp ../convertion_scripts/hbonds.tbl .<br />
cp ../convertion_scripts/aco.tbl .<br />
cp ufc1_1.mtf com/.</pre> <br />
&nbsp;&nbsp;&nbsp; 4.&nbsp; Modify input file <tt>rebuild.inp</tt> and then run <tt>cns &lt; rebuild.inp</tt> as described above. <br />
<br />
&nbsp;&nbsp;&nbsp; 5.&nbsp; Modify input file <tt>re_h2oc.inp</tt> as described above, remember to set <tt>$number_of_struct = 20</tt>. <br />
<br />
&nbsp;&nbsp;&nbsp; 6.&nbsp; Run macro <tt>subaba</tt> <br />
<br />
<code></code> <br />
<pre> #bash<br />
for i in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20<br />
do<br />
mkdir ref$i<br />
cd ref$i<br />
cp ../com/* .<br />
cp ../jan24/ufc1_rb_$i.pdb ufc1_rb_1.pdb<br />
qsub cns.sc<br />
cd ..<br />
<br />
donedone</pre> <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; where <tt>cns.sc</tt> is a PBS macro used to submit CNS job by using PBS: <br />
<pre> #!/bin/csh<br />
#PBS -m e<br />
#PBS -q short_c<br />
#PBS -l nodes=1:GM:ppn=1<br />
#PBS -l walltime=00:56:00<br />
#PBS -o out1<br />
#PBS -j oe<br />
#PBS -N clean1<br />
cd $PBS_O_WORKDIR<br />
echo "working directory = "$PBS_O_WORKDIR<br />
set NN = `cat $PBS_NODEFILE | wc -l`<br />
echo "NN = "$NN<br />
# Run Job<br />
cns &lt; re_h2oc.inp<br />
echo "ALL Done!"</pre> <br />
&nbsp;&nbsp;&nbsp; 7.&nbsp; Run <tt>getpdb</tt> to collect refined structures. <br />
<pre> #bash<br />
for i in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20<br />
do<br />
cd ref$i<br />
cp ufc1_ref_1.pdb ../pdb/ufc1_ref_$i.pdb<br />
cp ufc1_ref_1.vio ../pdb/ufc1_ref_$i.vio<br />
cd ..<br />
done</pre> <br />
&nbsp;&nbsp;&nbsp; 8. Run script [[Media:Agrupa.txt|Agrupa]] to put all 20 conformers into a single PDB file.<tt></tt> <br />
<pre> Agrupa ufc1_ref*.pdb &gt; ufc1all.pdb</pre> <br />
=== '''Running CNS for Proteins with Metal Ions''' ===<br />
<br />
The CNS refinement for proteins with metal ions can also be performed with the new WaterrefCNS script, using the appropriate metal ion nomenclature in both the pdb coordinates and distance constraints. <br />
<br />
For manual CNS refinement please do the refinement as following: <br />
<br />
1.&nbsp; Set environment for CNS1.1 by run source /farm/users/gliu/alias.cns <br />
<pre> alias cns1 /farm/software/cns/cns_solve_1.1/intel-i686-linux_g77/bin/cns<br />
setenv CNS_TOPPAR /farm/data/gliu/cns1/</pre> <br />
In addition to the topology and parameter files, the metal ion parameter file <tt>ion.top</tt> is required. An example can be found in "/farm/users/gliu/projects/cns_cuttha_cis" with all required input files. 2.&nbsp; Prepared required files as described above (final.tbl, final cns format PDB files and put in xplorPDB dir with name as sa_#.pdb) except the PDB file should include the metal ions with format according to [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/CNS CNS] library <tt>ion.top</tt>. cp sa_1.pdb as&nbsp; template.pdb, input files for creating mtf file Note that alignment is important. eg: <br />
<pre>ATOM 1249 OT2 ALA 83 69.296 13.232 5.744 1.00 0.00<br />
ATOM 1250 ZN+2 ZN2 150 63.086 13.789 -10.407 1.00 0.00 zinc</pre> <br />
3.&nbsp; Run generate_h2o.inp once to create temp_h2o.pdb and temp_h2o.mtf. The extra proton atom in the ligand residues, eg. HIS HD1 or CYS S, are removed by editing the generate_h2o.inp; cis proline is also defined here in the generate_h2o.inp (resid is the residue number prior the proline). <br />
<pre>{* any special prosthetic group patches can be applied here *}<br />
{===&gt;}<br />
delete select (name hg and resname cys and resid 61) end<br />
delete select (name hg and resname cys and resid 85) end<br />
delete select (name hd1 and resname his and resid 46) end<br />
delete select (name he2 and resname his and resid 83) end<br />
patch cisp<br />
reference=1=( resid 13 )<br />
end</pre> <br />
4.&nbsp; Edit generate_1.inp to remove the extra proton as did above. <br />
<br />
5.&nbsp; Run generate_20.com, this will run generate_#.inp 20 times, updating each pdb number and this creates cnsPDB/sa_cns_#.pdb <br />
<br />
6.&nbsp; Edit and run re_h2o_cu.inp, the refined pdb is kept in refinedPDB, or <br />
<br />
7.&nbsp; Use subcns to submit cns refinement by using PBS: eg, type "sh subcns". Before run subcns , make a folder " com" contains the following file. Type getpdb to get refined pdb files in refinedPDB after it finished. <br />
<br />
*cns.sc: PBS submission <br />
*cutc_h2o.mtf: mtf file created as descrive above <br />
*topology and parameter files: parallhdg5.3C.pro, parallhdg5.3.pro, topallhdg5.3.pro <br />
*re_h2o_cu.inp: input file for cns refinement<br><br />
<br />
<br />
=== '''Updated WaterRefCNS script at CABM for use with RDC constraints, metal ions, and dimers''' ===<br />
<br />
In 2011 the WaterRefCNS script was updated by Roberto Tejero.&nbsp; The current implementation uses CNS 1.3 and is capable of performing CNS&nbsp;water refinements including RDC&nbsp;constraints, metal ions constraints and on dimers. <br />
<br />
There are new defaults for the molecular dynamics steps.&nbsp; Again, at CABM&nbsp;we typically run calculations on master3.&nbsp; Here is the list of options and flags, and how the program is launched.&nbsp; Input files can be prepared using the <br />
<br />
PdbStat program. <br />
<pre> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ <br />
WaterRefCNS -- a tool to launch structure refinement with water<br />
--Roberto Tejero, 2011<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ <br />
<br />
Usage: /farm/software/WaterRefinement_cns/WaterRefCNS -na NamProt -que [PBS|No] -pro [xpl|cns] <br />
<br />
** Full set of options [not all are needed for a perfect run, use defaults] <br />
<br />
PROTEIN NAME <br />
-di for dimers <br />
-na NamProt Name of the protein (required!) <br />
-seed &lt;int&gt; Seed for random number generator (optional), (def. 31415) <br />
QUEUE SYSTEM AND PROTOCOL<br />
-pr [cns|xpl] Protocol to use, X-POR, CNS def cns <br />
-qu [PBS|NO] Que system to use, one of PBS or NOQUE def NOQUE<br />
PATCHES - SPECIAL RESIDUES <br />
-ci n1,n2,.. CIS pep info (PRO res. num) in comma separated list, i.e 56,89 <br />
-cysn n1,n2,.. CYHN pep (deprotonated SG CYS) res. num in comma separated list, i.e 10,65<br />
-hisd n1,n2,.. HISD pep info (HIS res num) in comma separated list, i.e 12,43 <br />
-hise n1,n2,.. HISE pep info (HIS res num) in comma separated list, i.e 25,67 <br />
CYANA CNS/X-PLOR <br />
~~~~~ ~~~~~~~~~~~ <br />
HIST HISE (HIS with no HD1) <br />
HIS HISD (HIS with no HE2) <br />
HIS+ HIS (HIS with both protons) <br />
<br />
-ss n1-n2,n3-n4 S-S bridge info in comma separated, dash-separated pairs, i.e. 2-24,30-40 <br />
MOLECULAR DYANMICS <br />
-av [center|sum] average for the distance restraints (for CNS, XPLOR) def sum <br />
-cool N number of cycles for cooling stage, default is 100 <br />
-heat N number of cycles in heating stage, default is 200 <br />
-hot N number of cycles in HOT stage, default is 1000 <br />
-par &lt;string&gt; nonbonded params, one of OPLSX|PARAM19|PARMALLH6|CONTACT (def. PARAM19)<br />
-sca N Scale terms (noe, dihe, hbond) by xN times (def. 1.0) <br />
-tsc &lt;real&gt; time step for cooling dynamics (def. 0.003) <br />
-tsh &lt;real&gt; time step for heating dynamics (def. 0.004) <br />
-tsr &lt;real&gt; time step for hot stage dynamics (def. 0.004) <br />
RDC DATA <br />
-DaR1 da,r Da and R for media 1, comma separated values -- overrides automatic detection <br />
-DaR2 da,r Da and R for media 2, comma separated values -- overrides automatic detection <br />
-wrdc1 &lt;real&gt; final weight --ksani1-- for RDC set 1 (def. 0.3) <br />
-wrdc2 &lt;real&gt; final weight --ksani2-- for RDC set 2 (def. 0.3) <br />
SYMMETRY <br />
-sym &lt;string&gt; Activates symmetry, string can be: all | 2:50,52:70 <br />
-wncsi &lt;real&gt; Initial weight for NCS symmetry statement (def 1.0) <br />
-wncsf &lt;real&gt; Final weight for NCS symmetry statement (def 20.0) <br />
HELP - INFO <br />
-he Display this help message <br />
-req Shows the requirements to run this script <br />
-res Displays a short guide to results (file BeSureToReadme) <br />
<br />
Examples: <br />
/farm/software/WaterRefinement_cns/WaterRefCNS -na RTT01 -di -que no -pr cns -ci 22 -hisd 12,42 -cysn 50,89 -wrdc1 1.5 -tsc 0.001 <br />
<br />
<br />
</pre> <br />
Some specific comments: <br />
<br />
1.&nbsp; For calculations using RDC constraints: by default the program uses the Da and R for each individual model in the ensemble.&nbsp; One can override this and enforce a "global" Da an R using the flag: <br />
<br />
"-DaR1 da,r".&nbsp; <br />
<br />
2.&nbsp; One can also manually adjust the relative weights of the RDCs from different alignments using the "-wrdc1" command.&nbsp; This is often useful when dealing with RDCs from PAG. <br />
<br />
3.&nbsp; Dimers are specified using the flag "-di". <br> <br />
<br />
== '''CNS Refinement with RDC constraints, metal ions using FMCGUI at Arrowsmith lab, UofT''' ==<br />
<br />
<font face="Arial, sans-serif"><font size="4">'''Step 1. Input files.'''</font></font> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">In order to perform water refinement using FMCGUI the following files should be prepared/available:</font></font> <br />
<br />
<br> <br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">structure file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">distance constraints file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">dihedral angles constraints file</font></font><br />
<br />
<br> <br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">User can include in the refinement the following additional constrains/data as well:</font></font> <br />
<br />
<br> <br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">hydrogen bond constraints file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data collected in different media; (up to 3 media are allowed)</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Zinc ions</font></font><br />
<br />
<br> <br> <br />
<br />
<br> <br />
<br />
#<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Structure file and constraints file format.</font></font><br><br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">It is expected that the structural, distance constraints, and dihedral angle constraints files are available as output of CYANA2.1 /CYANA3.0 structure calculations. The format of these files correspond to the format of</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">'final.pdb'</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">, 'f</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">inal.upl</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">', and '</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">final.aco'</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">ouput files. At the same time, these files could be loaded in FMCGUI in CNS/XPLOR format as well. It should be noted that structural file should contain key word MODEL that separate lines corresponding to different structural models.</font></font></font></font> <br />
<br />
<u>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Example.</font></font></font></font>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"></font></font></font></u><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">PDB file in CNS format:</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf20.jpeg]] <br />
<br />
&nbsp;<br> <br />
<br />
&nbsp; 2. <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data format</font></font><br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The RDC data can be used in CYANA or PALES formats. One can use RDC data collected in up to 3 media. All data from the same media should be included in the same file. When data are prepared in PALES format than a file should contain data only from one media. When using CYANA3 format, data from different media can be included in one file</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">(See Examples r1,and r2).</font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font><br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example r1.</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data in CYANA3 format.</font></font></font></font> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This example present file with HN and NC rdc's measured in 2 different media.</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf5.jpg]]<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example r2</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">. RDC data in PALES format.</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This example present file with HN, CCA, and NC rdc's measured in 1 media.</font></font></font></font> <br />
<br />
[[Image:Wrf6.jpg]] <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">&nbsp;3.&nbsp; Zinc ligands file format.</font></font><br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The Zinc-ligands file contains list of residues that coordinate each of Zinc ions present in the structure. There could be 3 or 4 Zinc ligands for one Zinc ion. Possible types of residues that coordinate Zinc ion are CYS, HIS, ASP, and GLU.</font></font>&nbsp; <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">(See Example z1)</font></font><br> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example z1</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">. Zinc- ligands file.</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font> <br />
<br />
[[Image:Wrf7.jpg]] <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This file indicate that there are 2 Zinc ions, one Zinc is coordinated by 4 residues 21, 26, 31, and 34 while the other Zinc is coordinated by 3 residues 48, 52, and 62</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This Zinc-file could be created using FMCGUI command [</font></font><font color="#993366"><font face="Times New Roman, serif"><font size="3">'''Structure&gt;Add ZN ligands'''</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">]:</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf8.jpg]]<br> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">User have to type IDs of residues that ligate zinc ion into the popped up entry window “ZN ligands” (see Figure). If there are a few zinc ions, information for each ion should be provided in a separate row.</font></font> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="4">'''Step 2. Setting up water refinement calculations using FMCGUI.'''</font></font> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">2.1<br />
Open new project [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;new'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]<br />
or load the old one [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;load'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]</span></font></font> <br />
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2.2&nbsp;&nbsp; <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">Select<br />
command</span></span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><br />
[</span></font></font>'''<font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Structure&gt;Water Refinement&gt;calculate</font></font></font>'''<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">]</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The popped up “Water Refinement” window where user is asked to select a number of files with coordinates and constraints (see Figure). The entry fields marked in read (PDB file, Distance constraints, and Dihedral angle constraints) should be specified while the others could be left blank. In addition to coordinates and constraints, user can indicate cisProline residues (if there are any) and to specify singly protonated HIS residues that are double protonated by default.</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">By pressing OK button in “water Refinement” window a new “Parameters” window</font></font> <br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">name of the water refinement directory</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">flag for distance constraints transformation (could be 1 or 2).&nbsp;</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">If the flag for distance constraints transformation is set to 2 then no change in the upper distance limit is performed. If the flag is set to 1 (default value), then the upper distance limits are modified by classifying each constraint in one of the 4 classes – strong, medium, weak and very weak</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">force constants for RDC constraints (if there are any) on the final stage of the refinement protocol.&nbsp;</span></font></font><br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Pressing OK button starts the setup of water refinement calculations using provided information the specified directory.</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The set up consists of the following operations:</font></font> <br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Splitting structure file in a number of separate files for each model. The atom names are changed from CYANA to CNS nomenclature.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming dihedral angle constraints from CYANA to CNS format. (If constraints are provided in CNS format then no transformation is performed).</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming distance NOE constraints from CYANA format to CNS format. This transformation includes changing atom names from CYANA to CNS nomenclature. If the flag for distance constraints transformation is set to 2 then no change in the upper distance limit is performed. If the flag is set to 1 (default value), then the upper distance limits are modified by classifying each constraint in one of the 4 classes – strong, medium, weak and very weak.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming Hbond constraints (if any) from CYANA format to CNS format</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Generating mtf-file for CNS calculations</font></font><br />
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*"Repairing" each input structure in accord with the mtf-file&nbsp;<br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Generating RDC constraints, a separate file for each media</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Estimating Da and R of alignment tensor for each media. This is performed by fitting RDC data to each model structure using program PALES, with subsequent averaging of the fitting results.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">Generating CNS script "re_h2o*_tmpl.inp" for the refinement calculations<br />
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<br> <font face="Arial, sans-serif"><font size="4">'''Step 3.&nbsp; Water refinement calculations using CNS.'''</font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">In a separate terminal, go to the refinement directory and run the nely generated python script<br />
“</span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">run_refine.py</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">”.</span></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This script will start N jobs in parallel, where N is the number of structural models to refine. The number of structures and the number of processor to use in the calculations can be specified by making modification of the top lines in script “</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">run_refine.py</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">”.<br />
If user want to reset the values for some refinement parameters, for<br />
example the values of Da and R, it could be done by modifying<br />
corresponding lines in the header of the CNS script<br />
</span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">“</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">re_h2o*_tmpl.inp</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”.<br />
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<font face="Arial, sans-serif"><font size="4">'''Step 4. Summarizing results of refinement using FMCGUI.'''</font></font> <br> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">Open<br />
new project [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;new'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]<br />
or load the old one [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;load'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]</span></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">Select<br />
command</span></span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"> </span></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">[</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Structure&gt;Water Refinement&gt;summary'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]&nbsp;&nbsp;&nbsp;&nbsp;</span></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">In the popped up “Water refinement summary” window user will be asked to specify residues used for structure superposition and to select the water refinement directory “WATDIR” were the refinement calculations were made.</font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font>[[Image:Wrf19.jpg]] <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The refined structural models will be superimpose and combined in one file. The created summary reports values of different energy components and constraint violation statistic for each structural model. A new directory “WATDIR_results” will be created, where WATDIR is the name of selected water refinement directory. The new directory contains final superimposed coordinates, distance and dihedral angle constrains in a format suitable for PDB deposition, summary and constraint violations reports, files</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">“</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">final_cns_summary</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”<br />
and “</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">final_violations</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”<br />
respectively. (See examples S1 and S2)</span></span></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">''<u><span style="font-weight: normal;">Example</span></u>'' <u>S1</u></span><u>.</u> File "final_cns_summary"&nbsp;</font></font></font></font></font></font></font></font></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The file shows the total energy, energy components, and number of constraints violations for each refined structure as well as corresponding average over all structures values.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<u>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">ExampleS2.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></u><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">File “</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">final_violations</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">”</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The file contains a list of all constraints violated in at least one structural model. For each constraint the the number of structural models that violate the&nbsp; constraint is shown.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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== <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>'''Files for Download'''</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> ==<br />
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#<u>[[Media:AtomtransC.tbl|atomtransC.tbl]]: atom name translation table</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Agrupa.txt|Agrupa]]: script for concatenating pdb coordinate files into one file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 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11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Topallhdg5.3.sol|topallhdg5.3.sol]]: CNS solvent topology file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, 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#<u>[[Media:Parallhdg5.3.sol|parallhdg5.3.sol]]: CNS solvent parameter file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Boxtyp20.pdb|boxtyp20.pdb]]: Water box</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Protein.link|protein.link]]: CNS peptide linkage definition file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Generate_water.cns|generate_water.cns]]: CNS protein solvation script</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 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11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Print_coorheader.cns|print_coorheader.cns]]: CNS script</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, 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#<u>[[Media:Cns_cabm.tar.gz|cns_cabm.tar.gz]]: CABM CNS refinement protocol</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:WaterRefinement_UB.tar.Z|WaterRefinement_UB.tar.Z]]: CNS water refinement package - SUNY Buffalo</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Cyana2cns.cya|cyana2cns.cya]]: CYANA script to convert coordinates and constrains to XPLOR/CNS format</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Cu_cPro_cns_example.tar.gz|Cu_cPro_cns_example.tar.gz]]: Example of CNS refinment for protein with Cu and cis-Pro</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Zn.tar|zn.tar]]: Exampleof CNS refinment for protein with zn (from Alex Lemark)</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Er317_2zn_CNSw_example.tar.gz|er317_2zn_CNSw_example.tar.gz]]: Example of CNS refinment for protein with zn (from Theresa Ramelot)</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
<br />
== <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>'''References'''</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> ==<br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>[http://www.ncbi.nlm.nih.gov/pubmed/9757107?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=14 1.&nbsp; Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. and Warren,G.L. (1998) Crystallography &amp; NMR system: a new software suite for macromolecular structure determination. ''Acta Crystallogr. D54'', 905-921.]</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u><br></u> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Structure_Refinement_Using_CNS_Energy_Minimization_With_Explicit_Water&diff=3843
Structure Refinement Using CNS Energy Minimization With Explicit Water
2011-10-05T15:18:04Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
Crystallography &amp; NMR System (CNS) is a program has been designed to provide a flexible multi-level hierachical approach for the most commonly used algorithms in macromolecular structure determination [1]. Functions include heavy atom searching, experimental phasing (including MAD and MIR), density modification, crystallographic refinement with maximum likelihood targets, and NMR structure calculation using NOEs, J-coupling, chemical shift, and dipolar coupling data. <br />
<br />
The latest version is 1.21. Please check the website [http://cns-online.org/ http://cns-online.org] for additional information. <br />
<br />
== '''CNS Refinement Protocol''' ==<br />
<br />
The final structure from CYANA should be refined using CNS energy minimization with explicit water before PDB deposition. <br />
<br />
The common input files are: <br />
<br />
*PDB coordinates <br />
*NOE constraints <br />
*Dihedral angle constraints <br />
*Hydrogen bond constraints<br />
<br />
==== '''Converting input files from CYANA to XPLOR/CNS''' ====<br />
<br />
If the final structure calculation was performed with CYANA 2.1, then the constraint and coordinate files must be first converted to XPLOR/CNS format. <br />
<br />
==== '''Using cyana2cns.cya script''' ====<br />
<br />
This procedure can be used to prepare input files to use with the WaterRefCNS script (see below). <br />
<br />
#Download [[Media:Cyana2cns.cya|cyana2cns.cya]] into directory with the final structure calculation. <br />
#Modify the <tt>read pdb</tt>, <tt>read upl</tt> and <tt>read aco</tt> lines if necessary. <br />
#Start CYANA 2.1 and run <tt>cyana2cns.cya</tt>.<br />
<br />
*All conformers will be stored in a single PDB file <tt>KKK.pdb</tt>. If you plan run water bath refinement manually, use the <tt>p2X</tt> program instead. <br />
*The resulting NOE constraint file <tt>KKK_noe.tbl</tt> will have all lower limits set to 0. If want them to be set according to VdW radii, use the <tt>r2X</tt> program instead. Experience shows that there is no significant effect on geometry and quality scores as reported by PSVS.<br />
<br />
==== '''Using programs p2X and r2X and d2X''' ====<br />
<br />
These programs were written by Alex Lemak at the University of Toronto. They are typically used to prepare input files for manual CNS water bath refinement. <br />
<br />
Go to <tt>~1_projects/targetID/structure/cns/</tt> directory and <tt>conversion_scripts</tt> sub directory, convert required input files to CNS format from CYANA format by using macro <tt>mkfil</tt>:&nbsp; <br />
<pre> p2X cyana2.pdb pref <br />
r2X found-c.upl noeupl.tbl <br />
r2X gr-hbonds.upl hbonds.tbl<br />
cat noeupl.tbl hbonds.tbl &gt; noe.tbl <br />
d2X found-c.aco aco.tbl <br />
</pre> <br />
*<tt>p2X splits conformers from a pdb file generated by CYANA 2.1 into individual pdb files (one file per conformer) and converts atom names to CNS format. The second argument "pref"</tt> specifies a common prefix name for the output pdb files. It should be no more than 4 characters long. <br />
*<tt>r2X</tt> converts distance constraints (noe/hbond) from CYANA 2.1 format to CNS format. The lower limits in the resulting constraint file are set according to VdW radii. <br />
**Distance constraints obtained from CYANA macro <tt>caliba</tt> or <tt>calibration</tt> can be used as input files. <br />
**Distance constraints obtained from CYANA macro <tt>peak calibrate</tt> should have the pseudo-atom correction run before used as input files, run following commands under CYANA: <br> <pre>read upl final.upl<br />
</pre><br />
<br />
distance correct write upl final_corrected.upl This, however, will not add corrections for multiplicity! <br />
<br />
*<tt>d2X</tt>, to run: <tt>d2X cyana2.aco cns.tbl</tt> This will convert angle constraints from CYANA format to CNS format. If you don't have any dihedral angle constrains, create an empty file.<br />
<br />
Both p2X and r2X require a translation table file [[Media:AtomtransC.tbl|atomtransC.tbl]]. <br />
<br />
If have used simplified pseudoatom names (H* instead of Q*) with option pseudo=2 in CYANA 2.1, you may first want to them before running p2X and r2X:<br> <br />
<pre>pseudo=0<br />
read upl noe.upl<br />
read pdb cyana2.pdb<br />
write upl out.upl<br />
write pdb out.pdb all<br />
</pre> <br />
Note that pseudo=0 should be set '''before''' loading PDB and constraint files. <br />
<br />
==== '''Conversion from DYANA''' ====<br />
<br />
Use CYANA macro <tt>MigrateFromDyanaCyana1.cya</tt> to convert of data files from standard CYANA1.x or DYANA nomenclature to the standard IUPAC nomenclature used by CYANA, then do then same thing as above. <br> <br />
<pre> translate dyana # use Cyana 1.x/Dyana nomenclature<br />
read seq demo-dyana.seq # read sequence from Cyana 1.x/Dyana<br />
read upl demo-dyana.upl unknown=warn # read upper distance limits from Cyana 1.x/Dyana<br />
read aco demo-dyana.aco unknown=warn # read angle restraints from Cyana 1.x/Dyana<br />
translate off # return to standard (IUPAC) nomenclature<br />
write demo-cyana.seq # save sequence in standard Cyana format<br />
write upl demo-cyana.upl # save upper distance limits in standard Cyana format<br />
write aco demo-cyana.aco # save angle restraints in standard Cyana format <br />
</pre> <br />
<br> <br />
<br />
=== '''Running CNS using WaterRefCNS script at CABM''' ===<br />
<br />
The WaterRefCNS script, developed at Rutgers University by Roberto Tejero, performs many of the above CNS water refinement protocols in an easy to use script with many user controled options. <br />
<br />
To see a list of options type the command: <br />
<pre> WaterRefCNS<br />
</pre> <br />
You will see a list of options and how the program is used. <br />
<pre> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
WaterRefCN -- a tool to launch structure refinement with water<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
<br />
Usage: /farm/software/WaterRefinement_cns/WaterRefCNS -na NamProt -que [PBS|No] -pro [xpl|cns]<br />
<br />
** Full set of options [not all are needed for a perfect run, use defaults]<br />
<br />
-na NamProt Name of the protein NOTE: this is mandatory, no default value <br />
-qu [PBS|NO] Que system to use, one of PBS or NOQUE def NOQUE<br />
-pr [cns|xpl] Protocol to use, X-POR, CNS def cns <br />
-av [center|sum] average for the distance restraints (for CNS, XPLOR) def sum <br />
-ci n1,n2 CIS pep info (PRO res num) in comma separated list, i.e 56,89 <br />
-ss n1-n2,n3-n4 S-S bridge info in comma separated, dash-separated pairs, i.e. 2-24,30-40 <br />
-he Display this help message<br />
-hisd n1,n2 HISD pep info (HIS res num) in comma separated list, i.e 12,43<br />
-hise n1,n2 HISE pep info (HIS res num) in comma separated list, i.e 25,67<br />
-heat N number of cycles in heating stage, default is 200 <br />
-hot N number of cycles in HOT stage, default is 1000 <br />
-cool N number of cycles for cooling stage, default is 100 <br />
-sc N Scale all terms (noe, dihe, hbond) by xN times def 1 <br />
-par string Choice of nonbonded params, one of OPLSX|PARAM19|PARMALLH6|PROLSQ|CONTACT<br />
default is OPLSX<br />
-seed &lt;int&gt; Seed for random number generator, default 31415 <br />
<br />
Examples:<br />
/farm/software/WaterRefinement_cns/WaterRefCNS -na SR358 -que no -pr cns -ci 22 <br />
<br />
</pre> <br />
Given a protein name KKK, here are the input files required to run WaterRefCNS: <br />
<br />
*&nbsp; pdb coordinates (in a single file) prepared in Xplor format using PdbStat.&nbsp; The name of the file should be:&nbsp; <tt>KKK.pdb</tt> <br />
*&nbsp; an noe.tbl file in CNS format.&nbsp; You can convert CYANA a&nbsp;final.upl file using PdbStat.&nbsp; We typically add 10-15% to the upper limit and make the lower limit vdw.&nbsp; The name of this file should be:&nbsp; <tt>KKK_noe.tbl</tt> <br />
*&nbsp; a dihe.tbl file in CNS&nbsp;format.&nbsp; Again, you can convert a CYANA .aco file using PdbStat.&nbsp; The name of this file should be:&nbsp; <tt>KKK_dihe.tbl</tt> <br />
*&nbsp; an hbond.tbl file in CNS format (optional).&nbsp; The name of this file should be:&nbsp; <tt>KKK_bhond.tbl</tt><br />
<br />
<br> <br />
<br />
In order to run WaterRefCNS on the CABM-NMR cluster: <br />
<br />
#Log into hummer or master3 <br />
#go to the directory with the above 4 files <br />
#type:<br />
<pre> /farm/software/WaterRefinement_cns/WaterRefCNS -na KKK -que PBS</pre> <br />
and that's it.&nbsp; If you have some CIS residue you add <tt>-ci</tt> NUMRES to the options above. If you want to have better vdw violation, you can add <tt>-par PARAM19</tt> to the options above. <br />
<br />
'''Future work:''' <br />
<br />
Currently, the WaterRefCNS script does not support refinement with RDC's.&nbsp; <br />
<br />
<br> <br />
<br />
=== '''WaterRefCNS at SUNY Buffalo''' ===<br />
<br />
The CABM water refinement package was customized to run on computers at UB: <br />
<br />
*Fixed a bug in <tt>WaterRefCNS</tt> so that the assembled refined PDB structure file has a <tt>.pdb</tt> extension. <br />
*PBS queue submission code in <tt>WaterRefCNS</tt> modified for use on U2 cluster <br />
*Added <tt>-hisd</tt> and <tt>-hise</tt> options to use neutral histidine isoprotomers. This required modification of <tt>WaterRefCNS</tt>, <tt>cns_refine_h2o.inp</tt> and <tt>generate_h2o.inp</tt>. <br />
*Fixed a bug in the protein topology file <tt>topallhdg5.3.pro</tt>. The line <tt>atom CD1 type=CR1E charge=0.130 end</tt> in the HISE patch section was removed. <br />
*Fixed a bug that caused the order of conformers to be changed in the output PDB file by replacing the line <tt>$WATREFLIB/Agrupa *.pdb &gt; All_${Name}_cns.pdb</tt> with <tt>$WATREFLIB/Agrupa `ls resa_?.pdb&nbsp;; ls resa_??.pdb` &gt; All_${Name}_cns.pdb</tt> in the <tt>WaterRefCNS</tt> script.<br />
<br />
The modified package is installed in <tt>/nsm/chem/cen2/HTP2/3_src/WaterRefinement_cns</tt> on the local server and in <tt>/san/projects1/szypersk/src/WaterRefinement_cns</tt> on the U2 Linux cluster. Make sure that this directories is included in your path on <tt>spins*</tt> workstations and U2, respectively. <br />
<br />
For reference, the download location is here: [[Media:WaterRefinement_UB.tar.Z|WaterRefinement_UB.tar.Z]] <br />
<br />
The required files are: <br />
<br />
*PDB coordinates (with atom names in XPLOR/CNS format) <br />
*NOE constraints (in XPLOR/CNS format) <br />
*Dihedral angle constraints (in XPLOR/CNS format) <br />
*Hydrogen bond constraints (in XPLOR/CNS format)<br />
<br />
Assuming that your coordinate file is called <tt>KKK.pdb</tt>, the constraint files should be named <tt>KKK_noe.tbl</tt>, <tt>KKK_dihe.tbl</tt> and <tt>KKK_hbond.tbl</tt>. If you don't have dihedral angle and/or hydrogen bond constraints, then create the corresponding empty files. <br />
<br />
You may need to add a line like <tt>nassign=1000</tt> at the top of the constraint files. The value should be equal to or greater than the number of constraints in the file. <br />
<br />
WaterRefCNS assumes "sum of r<sup>-6</sup>" averaging by default. When using "center"-averaged UPLs (e.g. from <tt>caliba</tt>) add <tt>-av center</tt> when starting WaterRefCNS. For information on averaging conventions for calibration in CYANA see [[NOE Calibration Using CYANA|NOE Calibration in CYANA]] <br />
<br />
It is recommended to run it on U2 Linux cluster. Examples: <br />
<br />
#<pre>WaterRefCNS -na KKK -que PBS</pre> <br />
#<pre>WaterRefCNS -na KKK -que PBS -ci 21,49</pre>residues 21 and 49 are cis-Pro <br />
#<pre>WaterRefCNS -na KKK -que PBS -hise 40,82 -hisd 32,65</pre>residues 40 and 82 are ε-protonated neutral His <br> residues 32 and 65 are δ-protonated neutral His <br />
#<pre>WaterRefCNS -na KKK -que PBS -av center </pre>for use with center-averaged calibration<br />
<br />
To run on <tt>spins*</tt> Linux workstations without queue system use <tt>-que NO</tt> option instead of <tt>-que PBS</tt> option. The default is no queue system, so you can omit the <tt>-que</tt> option altogether. <br />
<br />
==== '''Running WaterRefCNS''' ====<br />
<br />
#Convert the coordinate and constraint files as described above. <br />
#Copy the converted files into a CNS working directory on the U2 cluster or a workstation (e.g. <tt>structure/cns/calc1</tt>). <br />
#Run the <tt>WaterRefCNS</tt> with the proper arguments. <br />
#If the calculation was successful, the refined coordinate file will be stored in the <tt>refinedPDB</tt> subdirectory. Check the <tt>BeSureToREADME</tt> for details.<br />
<br />
=== '''Running CNS "manually"''' ===<br />
<br />
Go to <tt>~1_projects/targetID/structure/cns/</tt> directory and <tt>cns_scripts</tt> sub directory, follow the procedures as describe below. <br />
<br />
'''1.&nbsp; Generating MTF topology file from a PDB file. '''<br>&nbsp;&nbsp;&nbsp; modify the header of the input file <tt>generate_mtf.inp</tt> and then run <tt>cns &lt; generate_mtf.inp &gt; mtf.log</tt>. <br />
<br />
*Modify input files path and name base on individual target as follows:<br />
<pre> ...<br />
{in} pdb_file="/san/user/gliu2/u2/cns/cns/convertion_scripts/ufc1_1.pdb";<br />
{in} param_file="/san/user/gliu2/u2/cns/cns/cns_scripts/parallhdg5.3C.pro";<br />
{in} topol_file="/san/user/gliu2/u2/cns/cns/cns_scripts/topallhdg5.3.pro";<br />
{in} plink_file="/san/user/gliu2/u2/cns/cns/cns_scripts/protein.link";<br />
{out} struct_file="ufc1_1.mtf"; <br />
...<br />
</pre> <br />
*Requires these files: <br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file <br />
*[[Media:Protein.link|protein.link]]: CNS peptide linkage definition file <br />
*For <tt>cis-Proline</tt>, define the residue Id of the residue prior to <tt>cis-Proline</tt> by additional lines in <tt>generate_mtf.inp</tt> before the WRITE command<br />
<pre> patch cisp<br />
reference=nil=( resid 90 )<br />
end <br />
</pre> <br />
*If you get an error message on the atom type, you also can try&nbsp;<br />
<pre> patch cipp<br />
reference=nil=( resid 90 )<br />
end </pre> <br />
*For neutral Nε- and Nδ-protonated histidines add these lines in <tt>generate_mtf.inp</tt> before the WRITE command<br />
<pre> patch hise<br />
reference=nil=( resid 40 )<br />
end<br />
patch hisd<br />
reference=nil=( resid 65 )<br />
end </pre> <br />
*For dimer refinement, add a line containing only <tt>TER</tt> between two monomer unit coordinates in the PDB conformer that is defined in <tt>generate_mtf_cis.inp</tt> as input file.<br />
<br />
'''<br>''' <br />
<br />
'''2.&nbsp; Rebuilding hydrogen atom positions for each structure. '''<br>&nbsp;&nbsp;&nbsp; Modify input file <tt>rebuild.inp</tt> and then run <tt>cns &lt; rebuild.inp &gt; rebuild.log</tt> <br />
<br />
*Modify input files path and name base on induvidual target as follows:&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;<br />
<pre> ...<br />
evaluate ($mtf_file="ufc1_1.mtf")<br />
evaluate ($pdbname_in="../convertion_scripts/ufc1")<br />
evaluate ($pdbname_out="ufc1_rb")<br />
evaluate ($number_of_struct= 20 )<br />
...<br />
evaluate ($topol_p_file="/san/user/gliu2/u2/cns/cns/cns_scripts/topallhdg5.3.pro")<br />
evaluate ($param_p_file="/san/user/gliu2/u2/cns/cns/cns_scripts/parallhdg5.3C.pro")<br />
...<br />
</pre> <br />
*Requires these files: <br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file<br />
<br />
'''3. Refining structures with explicit water.''' <br />
<br />
*Running <tt>CNS</tt> on workstation with single processor, modify input file <tt>re_h2oc.inp</tt> and then run <tt>cns &lt; re_h2oc.inp &gt; h2o.log</tt> <br />
*Modify input files path and name base on induvidual target as follows:<br />
<pre> ...<br />
evaluate ($mtf_file="ufc1_1.mtf")<br />
evaluate ($noe_file="../noe.tbl")<br />
evaluate ($dihe_file="../aco.tbl")<br />
evaluate ($hb_file="../hbond.tbl")<br />
evaluate ($pdbname_in="ufc1_rb")<br />
evaluate ($pdbname_out="ufc1_ref")<br />
evaluate ($number_of_struct= 20 )<br />
...</pre> <br />
*Modify the weight of energy contribution part if necessary, e.g. use 2x to 10x of the following scales:<br />
<pre> scale ambi 50<br />
scale dist 50<br />
scale hbond 50<br />
end<br />
restraints dihedral<br />
scale=200<br />
end</pre> <br />
*Requires <br />
**[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
**[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file <br />
**[[Media:Topallhdg5.3.sol|topallhdg5.3.sol]]: CNS solvent topology file <br />
**[[Media:Parallhdg5.3.sol|parallhdg5.3.sol]]: CNS solvent parameter file <br />
**[[Media:Boxtyp20.pdb|boxtyp20.pdb]]: Water box <br />
**[[Media:Generate_water.cns|generate_water.cns]]: CNS protein solvation script <br />
**[[Media:Print_coorheader.cns|print_coorheader.cns]]: CNS script <br />
*Run CNS on workstation with multiple processors. <br> CNS will refine each conformer independently and in parallel, it will be much faster if 20 conformer were refined in parallal at u2.ccr.buffalo.edu.<br />
<br />
&nbsp;&nbsp;&nbsp; 1.&nbsp; Run cns_convertion as descibed above &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp; 2.&nbsp; Go to cns_scripts directory, modify input file <tt>generate_mtf_cis.inp</tt> and then run =cns &lt; generate_mtf_cis.inp = as described above &nbsp;&nbsp;&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp; 3.&nbsp; Run macro <tt>getfil</tt> <br />
<pre> cp ../convertion_scripts/noe.tbl .<br />
cp ../convertion_scripts/hbonds.tbl .<br />
cp ../convertion_scripts/aco.tbl .<br />
cp ufc1_1.mtf com/.</pre> <br />
&nbsp;&nbsp;&nbsp; 4.&nbsp; Modify input file <tt>rebuild.inp</tt> and then run <tt>cns &lt; rebuild.inp</tt> as described above. <br />
<br />
&nbsp;&nbsp;&nbsp; 5.&nbsp; Modify input file <tt>re_h2oc.inp</tt> as described above, remember to set <tt>$number_of_struct = 20</tt>. <br />
<br />
&nbsp;&nbsp;&nbsp; 6.&nbsp; Run macro <tt>subaba</tt> <br />
<br />
<code></code> <br />
<pre> #bash<br />
for i in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20<br />
do<br />
mkdir ref$i<br />
cd ref$i<br />
cp ../com/* .<br />
cp ../jan24/ufc1_rb_$i.pdb ufc1_rb_1.pdb<br />
qsub cns.sc<br />
cd ..<br />
<br />
donedone</pre> <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; where <tt>cns.sc</tt> is a PBS macro used to submit CNS job by using PBS: <br />
<pre> #!/bin/csh<br />
#PBS -m e<br />
#PBS -q short_c<br />
#PBS -l nodes=1:GM:ppn=1<br />
#PBS -l walltime=00:56:00<br />
#PBS -o out1<br />
#PBS -j oe<br />
#PBS -N clean1<br />
cd $PBS_O_WORKDIR<br />
echo "working directory = "$PBS_O_WORKDIR<br />
set NN = `cat $PBS_NODEFILE | wc -l`<br />
echo "NN = "$NN<br />
# Run Job<br />
cns &lt; re_h2oc.inp<br />
echo "ALL Done!"</pre> <br />
&nbsp;&nbsp;&nbsp; 7.&nbsp; Run <tt>getpdb</tt> to collect refined structures. <br />
<pre> #bash<br />
for i in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20<br />
do<br />
cd ref$i<br />
cp ufc1_ref_1.pdb ../pdb/ufc1_ref_$i.pdb<br />
cp ufc1_ref_1.vio ../pdb/ufc1_ref_$i.vio<br />
cd ..<br />
done</pre> <br />
&nbsp;&nbsp;&nbsp; 8. Run script [[Media:Agrupa.txt|Agrupa]] to put all 20 conformers into a single PDB file.<tt></tt> <br />
<pre> Agrupa ufc1_ref*.pdb &gt; ufc1all.pdb</pre> <br />
=== '''Running CNS for Proteins with Metal Ions''' ===<br />
<br />
The CNS refinement for proteins with metal ions can also be performed with the new WaterrefCNS script, using the appropriate metal ion nomenclature in both the pdb coordinates and distance constraints. <br />
<br />
For manual CNS refinement please do the refinement as following: <br />
<br />
1.&nbsp; Set environment for CNS1.1 by run source /farm/users/gliu/alias.cns <br />
<pre> alias cns1 /farm/software/cns/cns_solve_1.1/intel-i686-linux_g77/bin/cns<br />
setenv CNS_TOPPAR /farm/data/gliu/cns1/</pre> <br />
In addition to the topology and parameter files, the metal ion parameter file <tt>ion.top</tt> is required. An example can be found in "/farm/users/gliu/projects/cns_cuttha_cis" with all required input files. 2.&nbsp; Prepared required files as described above (final.tbl, final cns format PDB files and put in xplorPDB dir with name as sa_#.pdb) except the PDB file should include the metal ions with format according to [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/CNS CNS] library <tt>ion.top</tt>. cp sa_1.pdb as&nbsp; template.pdb, input files for creating mtf file Note that alignment is important. eg: <br />
<pre>ATOM 1249 OT2 ALA 83 69.296 13.232 5.744 1.00 0.00<br />
ATOM 1250 ZN+2 ZN2 150 63.086 13.789 -10.407 1.00 0.00 zinc</pre> <br />
3.&nbsp; Run generate_h2o.inp once to create temp_h2o.pdb and temp_h2o.mtf. The extra proton atom in the ligand residues, eg. HIS HD1 or CYS S, are removed by editing the generate_h2o.inp; cis proline is also defined here in the generate_h2o.inp (resid is the residue number prior the proline). <br />
<pre>{* any special prosthetic group patches can be applied here *}<br />
{===&gt;}<br />
delete select (name hg and resname cys and resid 61) end<br />
delete select (name hg and resname cys and resid 85) end<br />
delete select (name hd1 and resname his and resid 46) end<br />
delete select (name he2 and resname his and resid 83) end<br />
patch cisp<br />
reference=1=( resid 13 )<br />
end</pre> <br />
4.&nbsp; Edit generate_1.inp to remove the extra proton as did above. <br />
<br />
5.&nbsp; Run generate_20.com, this will run generate_#.inp 20 times, updating each pdb number and this creates cnsPDB/sa_cns_#.pdb <br />
<br />
6.&nbsp; Edit and run re_h2o_cu.inp, the refined pdb is kept in refinedPDB, or <br />
<br />
7.&nbsp; Use subcns to submit cns refinement by using PBS: eg, type "sh subcns". Before run subcns , make a folder " com" contains the following file. Type getpdb to get refined pdb files in refinedPDB after it finished. <br />
<br />
*cns.sc: PBS submission <br />
*cutc_h2o.mtf: mtf file created as descrive above <br />
*topology and parameter files: parallhdg5.3C.pro, parallhdg5.3.pro, topallhdg5.3.pro <br />
*re_h2o_cu.inp: input file for cns refinement<br><br />
<br />
=== ===<br />
<br />
=== '''Updated WaterRefCNS script at CABM for use with RDC constraints, metal ions, and dimers''' ===<br />
<br />
In 2011 the WaterRefCNS script was updated by Roberto Tejero.&nbsp; The current implementation uses CNS 1.3 and is capable of performing CNS&nbsp;water refinements including RDC&nbsp;constraints, metal ions constraints and on dimers. <br />
<br />
There are new defaults for the molecular dynamics steps.&nbsp; Again, at CABM&nbsp;we typically run calculations on master3.&nbsp; Here is the list of options and flags, and how the program is launched.&nbsp; Input files can be prepared using the <br />
<br />
PdbStat program.<br />
<pre> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ <br />
WaterRefCNS -- a tool to launch structure refinement with water<br />
--Roberto Tejero, 2011<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ <br />
<br />
Usage: /farm/software/WaterRefinement_cns/WaterRefCNS -na NamProt -que [PBS|No] -pro [xpl|cns] <br />
<br />
** Full set of options [not all are needed for a perfect run, use defaults] <br />
<br />
PROTEIN NAME <br />
-di for dimers <br />
-na NamProt Name of the protein (required!) <br />
-seed &lt;int&gt; Seed for random number generator (optional), (def. 31415) <br />
QUEUE SYSTEM AND PROTOCOL<br />
-pr [cns|xpl] Protocol to use, X-POR, CNS def cns <br />
-qu [PBS|NO] Que system to use, one of PBS or NOQUE def NOQUE<br />
PATCHES - SPECIAL RESIDUES <br />
-ci n1,n2,.. CIS pep info (PRO res. num) in comma separated list, i.e 56,89 <br />
-cysn n1,n2,.. CYHN pep (deprotonated SG CYS) res. num in comma separated list, i.e 10,65<br />
-hisd n1,n2,.. HISD pep info (HIS res num) in comma separated list, i.e 12,43 <br />
-hise n1,n2,.. HISE pep info (HIS res num) in comma separated list, i.e 25,67 <br />
CYANA CNS/X-PLOR <br />
~~~~~ ~~~~~~~~~~~ <br />
HIST HISE (HIS with no HD1) <br />
HIS HISD (HIS with no HE2) <br />
HIS+ HIS (HIS with both protons) <br />
<br />
-ss n1-n2,n3-n4 S-S bridge info in comma separated, dash-separated pairs, i.e. 2-24,30-40 <br />
MOLECULAR DYANMICS <br />
-av [center|sum] average for the distance restraints (for CNS, XPLOR) def sum <br />
-cool N number of cycles for cooling stage, default is 100 <br />
-heat N number of cycles in heating stage, default is 200 <br />
-hot N number of cycles in HOT stage, default is 1000 <br />
-par &lt;string&gt; nonbonded params, one of OPLSX|PARAM19|PARMALLH6|CONTACT (def. PARAM19)<br />
-sca N Scale terms (noe, dihe, hbond) by xN times (def. 1.0) <br />
-tsc &lt;real&gt; time step for cooling dynamics (def. 0.003) <br />
-tsh &lt;real&gt; time step for heating dynamics (def. 0.004) <br />
-tsr &lt;real&gt; time step for hot stage dynamics (def. 0.004) <br />
RDC DATA <br />
-DaR1 da,r Da and R for media 1, comma separated values -- overrides automatic detection <br />
-DaR2 da,r Da and R for media 2, comma separated values -- overrides automatic detection <br />
-wrdc1 &lt;real&gt; final weight --ksani1-- for RDC set 1 (def. 0.3) <br />
-wrdc2 &lt;real&gt; final weight --ksani2-- for RDC set 2 (def. 0.3) <br />
SYMMETRY <br />
-sym &lt;string&gt; Activates symmetry, string can be: all | 2:50,52:70 <br />
-wncsi &lt;real&gt; Initial weight for NCS symmetry statement (def 1.0) <br />
-wncsf &lt;real&gt; Final weight for NCS symmetry statement (def 20.0) <br />
HELP - INFO <br />
-he Display this help message <br />
-req Shows the requirements to run this script <br />
-res Displays a short guide to results (file BeSureToReadme) <br />
<br />
Examples: <br />
/farm/software/WaterRefinement_cns/WaterRefCNS -na RTT01 -di -que no -pr cns -ci 22 -hisd 12,42 -cysn 50,89 -wrdc1 1.5 -tsc 0.001 <br />
<br />
<br />
</pre><br />
Some specific comments:<br />
<br />
1.&nbsp; For calculations using RDC constraints: by default the program uses the Da and R for each individual model in the ensemble.&nbsp; One can override this and enforce a "global" Da an R using the flag: <br />
<br />
"-DaR1 da,r".&nbsp; <br />
<br />
2.&nbsp; One can also manually adjust the relative weights of the RDCs from different alignments using the "-wrdc1" command.&nbsp; This is often useful when dealing with RDCs from PAG.<br />
<br />
3.&nbsp; Dimers are specified using the flag "-di".<br />
<br><br />
== '''CNS Refinement with RDC constraints, metal ions using FMCGUI at Arrowsmith lab, UofT''' ==<br />
<br />
<font face="Arial, sans-serif"><font size="4">'''Step 1. Input files.'''</font></font> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">In order to perform water refinement using FMCGUI the following files should be prepared/available:</font></font> <br />
<br />
<br> <br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">structure file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">distance constraints file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">dihedral angles constraints file</font></font><br />
<br />
<br> <br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">User can include in the refinement the following additional constrains/data as well:</font></font> <br />
<br />
<br> <br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">hydrogen bond constraints file</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data collected in different media; (up to 3 media are allowed)</font></font><br />
<br />
*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Zinc ions</font></font><br />
<br />
<br> <br> <br />
<br />
<br> <br />
<br />
#<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Structure file and constraints file format.</font></font><br><br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">It is expected that the structural, distance constraints, and dihedral angle constraints files are available as output of CYANA2.1 /CYANA3.0 structure calculations. The format of these files correspond to the format of</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">'final.pdb'</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">, 'f</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">inal.upl</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">', and '</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">final.aco'</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">ouput files. At the same time, these files could be loaded in FMCGUI in CNS/XPLOR format as well. It should be noted that structural file should contain key word MODEL that separate lines corresponding to different structural models.</font></font></font></font> <br />
<br />
<u>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Example.</font></font></font></font>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"></font></font></font></u><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">PDB file in CNS format:</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf20.jpeg]] <br />
<br />
&nbsp;<br> <br />
<br />
&nbsp; 2. <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data format</font></font><br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The RDC data can be used in CYANA or PALES formats. One can use RDC data collected in up to 3 media. All data from the same media should be included in the same file. When data are prepared in PALES format than a file should contain data only from one media. When using CYANA3 format, data from different media can be included in one file</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">(See Examples r1,and r2).</font></font> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font><br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example r1.</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">RDC data in CYANA3 format.</font></font></font></font> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This example present file with HN and NC rdc's measured in 2 different media.</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf5.jpg]]<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example r2</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">. RDC data in PALES format.</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This example present file with HN, CCA, and NC rdc's measured in 1 media.</font></font></font></font> <br />
<br />
[[Image:Wrf6.jpg]] <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">&nbsp;3.&nbsp; Zinc ligands file format.</font></font><br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The Zinc-ligands file contains list of residues that coordinate each of Zinc ions present in the structure. There could be 3 or 4 Zinc ligands for one Zinc ion. Possible types of residues that coordinate Zinc ion are CYS, HIS, ASP, and GLU.</font></font>&nbsp; <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">(See Example z1)</font></font><br> <br />
<br />
<br> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">''<u>Example z1</u>''</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">. Zinc- ligands file.</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font> <br />
<br />
[[Image:Wrf7.jpg]] <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This file indicate that there are 2 Zinc ions, one Zinc is coordinated by 4 residues 21, 26, 31, and 34 while the other Zinc is coordinated by 3 residues 48, 52, and 62</font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This Zinc-file could be created using FMCGUI command [</font></font><font color="#993366"><font face="Times New Roman, serif"><font size="3">'''Structure&gt;Add ZN ligands'''</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">]:</font></font></font></font> <br />
<br />
<br> <br />
<br />
[[Image:Wrf8.jpg]]<br> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">User have to type IDs of residues that ligate zinc ion into the popped up entry window “ZN ligands” (see Figure). If there are a few zinc ions, information for each ion should be provided in a separate row.</font></font> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<font face="Arial, sans-serif"><font size="4">'''Step 2. Setting up water refinement calculations using FMCGUI.'''</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">2.1<br />
Open new project [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;new'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]<br />
or load the old one [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;load'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]</span></font></font> <br />
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2.2&nbsp;&nbsp; <font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">Select<br />
command</span></span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><br />
[</span></font></font>'''<font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Structure&gt;Water Refinement&gt;calculate</font></font></font>'''<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">]</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The popped up “Water Refinement” window where user is asked to select a number of files with coordinates and constraints (see Figure). The entry fields marked in read (PDB file, Distance constraints, and Dihedral angle constraints) should be specified while the others could be left blank. In addition to coordinates and constraints, user can indicate cisProline residues (if there are any) and to specify singly protonated HIS residues that are double protonated by default.</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">By pressing OK button in “water Refinement” window a new “Parameters” window</font></font> <br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">name of the water refinement directory</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">flag for distance constraints transformation (could be 1 or 2).&nbsp;</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">If the flag for distance constraints transformation is set to 2 then no change in the upper distance limit is performed. If the flag is set to 1 (default value), then the upper distance limits are modified by classifying each constraint in one of the 4 classes – strong, medium, weak and very weak</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">force constants for RDC constraints (if there are any) on the final stage of the refinement protocol.&nbsp;</span></font></font><br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Pressing OK button starts the setup of water refinement calculations using provided information the specified directory.</font></font> <br />
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<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The set up consists of the following operations:</font></font> <br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Splitting structure file in a number of separate files for each model. The atom names are changed from CYANA to CNS nomenclature.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming dihedral angle constraints from CYANA to CNS format. (If constraints are provided in CNS format then no transformation is performed).</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming distance NOE constraints from CYANA format to CNS format. This transformation includes changing atom names from CYANA to CNS nomenclature. If the flag for distance constraints transformation is set to 2 then no change in the upper distance limit is performed. If the flag is set to 1 (default value), then the upper distance limits are modified by classifying each constraint in one of the 4 classes – strong, medium, weak and very weak.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Transforming Hbond constraints (if any) from CYANA format to CNS format</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Generating mtf-file for CNS calculations</font></font><br />
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*"Repairing" each input structure in accord with the mtf-file&nbsp;<br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Generating RDC constraints, a separate file for each media</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">Estimating Da and R of alignment tensor for each media. This is performed by fitting RDC data to each model structure using program PALES, with subsequent averaging of the fitting results.</font></font><br />
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*<font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">Generating CNS script "re_h2o*_tmpl.inp" for the refinement calculations<br />
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<br> <font face="Arial, sans-serif"><font size="4">'''Step 3.&nbsp; Water refinement calculations using CNS.'''</font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">In a separate terminal, go to the refinement directory and run the nely generated python script<br />
“</span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">run_refine.py</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">”.</span></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">This script will start N jobs in parallel, where N is the number of structural models to refine. The number of structures and the number of processor to use in the calculations can be specified by making modification of the top lines in script “</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">run_refine.py</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span style="font-weight: normal;">”.<br />
If user want to reset the values for some refinement parameters, for<br />
example the values of Da and R, it could be done by modifying<br />
corresponding lines in the header of the CNS script<br />
</span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">“</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">re_h2o*_tmpl.inp</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”.<br />
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<font face="Arial, sans-serif"><font size="4">'''Step 4. Summarizing results of refinement using FMCGUI.'''</font></font> <br> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">Open<br />
new project [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;new'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]<br />
or load the old one [</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Project&gt;load'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]</span></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">Select<br />
command</span></span></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"> </span></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">[</span></font></font><font color="#993366"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">'''Structure&gt;Water Refinement&gt;summary'''</span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">]&nbsp;&nbsp;&nbsp;&nbsp;</span></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">In the popped up “Water refinement summary” window user will be asked to specify residues used for structure superposition and to select the water refinement directory “WATDIR” were the refinement calculations were made.</font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font>[[Image:Wrf19.jpg]] <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The refined structural models will be superimpose and combined in one file. The created summary reports values of different energy components and constraint violation statistic for each structural model. A new directory “WATDIR_results” will be created, where WATDIR is the name of selected water refinement directory. The new directory contains final superimposed coordinates, distance and dihedral angle constrains in a format suitable for PDB deposition, summary and constraint violations reports, files</font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">“</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">final_cns_summary</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”<br />
and “</span></span></font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">final_violations</span></span></font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US"><span style="font-weight: normal;">”<br />
respectively. (See examples S1 and S2)</span></span></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><span lang="en-US">''<u><span style="font-weight: normal;">Example</span></u>'' <u>S1</u></span><u>.</u> File "final_cns_summary"&nbsp;</font></font></font></font></font></font></font></font></font></font></font></font></font></font><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The file shows the total energy, energy components, and number of constraints violations for each refined structure as well as corresponding average over all structures values.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>[[Image:Wrf12.jpg]]<br></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u><br></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<u>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">ExampleS2.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font>''<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></u><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">File “</font></font><font color="#0000ff"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">final_violations</font></font></font><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">”</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;">The file contains a list of all constraints violated in at least one structural model. For each constraint the the number of structural models that violate the&nbsp; constraint is shown.</font></font></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>[[Image:Wrf11.jpg]]<br></u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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== <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>'''Files for Download'''</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> ==<br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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#<u>[[Media:AtomtransC.tbl|atomtransC.tbl]]: atom name translation table</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Agrupa.txt|Agrupa]]: script for concatenating pdb coordinate files into one file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier 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11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
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#<u>[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, 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#<u>[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Topallhdg5.3.sol|topallhdg5.3.sol]]: CNS solvent topology file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
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#<u>[[Media:Parallhdg5.3.sol|parallhdg5.3.sol]]: CNS solvent parameter file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Boxtyp20.pdb|boxtyp20.pdb]]: Water box</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Protein.link|protein.link]]: CNS peptide linkage definition file</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Generate_water.cns|generate_water.cns]]: CNS protein solvation script</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Print_coorheader.cns|print_coorheader.cns]]: CNS script</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Cns_cabm.tar.gz|cns_cabm.tar.gz]]: CABM CNS refinement protocol</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:WaterRefinement_UB.tar.Z|WaterRefinement_UB.tar.Z]]: CNS water refinement package - SUNY Buffalo</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Cyana2cns.cya|cyana2cns.cya]]: CYANA script to convert coordinates and constrains to XPLOR/CNS format</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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#<u>[[Media:Cu_cPro_cns_example.tar.gz|Cu_cPro_cns_example.tar.gz]]: Example of CNS refinment for protein with Cu and cis-Pro</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
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#<u>[[Media:Zn.tar|zn.tar]]: Exampleof CNS refinment for protein with zn (from Alex Lemark)</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"></font></font></font></font></font></font></font></font></font></font></font></font></font></font><br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><br />
#<u>[[Media:Er317_2zn_CNSw_example.tar.gz|er317_2zn_CNSw_example.tar.gz]]: Example of CNS refinment for protein with zn (from Theresa Ramelot)</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
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== <font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>'''References'''</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> ==<br />
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<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u>[http://www.ncbi.nlm.nih.gov/pubmed/9757107?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=14 1.&nbsp; Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. and Warren,G.L. (1998) Crystallography &amp; NMR system: a new software suite for macromolecular structure determination. ''Acta Crystallogr. D54'', 905-921.]</u></font></font></font></font></font></font></font></font></font></font></font></font></font></font> <br />
<br />
<font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="4" style="font-size: 16pt;"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font color="#993366"><font face="Arial, sans-serif"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><font face="Courier New, monospace"><font size="2"><font face="Arial, sans-serif"><font size="2" style="font-size: 11pt;"><u><br></u> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font> </font></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=PdbStat&diff=3842
PdbStat
2011-10-05T14:24:34Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
The PdbStat program, written and actively developed by our colleague Roberto Tejero (Universita de Valencia), is routinely used in the Montelione lab for the analysis, conversion, and manipulation of coordinate and constraint files for protein structure determination. The program is also an integral component of the Protein Structure Validation Software package [1] used across the NESG consortium. <br />
<br />
The current version of the software is PdbStat 5.1 (July, 2008). There is no separate publication for PdbStat; we cite this program using Bhattacharya et al, 2007 [1]. Also, there is no official manual for the program. Below are a number of basic stand-alone PdbStat commands and common uses. Going forward, we can continue to add to this document as more applications are developed. <br />
<br />
Anyone interested in obtaining the latest version of PdbStat can contact Roberto at: [mailto:roberto.tejero@uv.es roberto.tejero@uv.es] <br> <br />
<br />
== '''Commands and Applications''' ==<br />
<br />
==== Starting the program ====<br />
<br />
<tt>pdbstat</tt><br />
<br />
==== Menu of Commands ====<br />
<br />
<tt>menu</tt> gives a list of commands and keywords recognized by PdbStat (also get this using the command: <tt>help command</tt>) <br />
<br />
Here are the recognized PdbStat commands for version 5.1: <br />
<pre>PdbStat&gt; menu<br />
<br />
Commands/Keywords currently recognized by PDBSTAT<br />
<br />
The commands/keywords currently recognized by PDBSTAT are given below. <br />
Type "help &lt;command&gt;" for more information on each PDBSTAT function.<br />
<br />
align analyze author bye class<br />
clear close chain check chiral<br />
delete debug dump energy evaluate <br />
find fit fix get help<br />
history hydrogen hyper kabsch ident/chain<br />
info initialize log/change load menu<br />
missing order phi quit rama<br />
read relax reset restore rotate<br />
save see set show rmsd<br />
to trans version what write<br />
<br />
homology commands: <br />
get change homology homa<br />
<br />
Other kind/commands/keywords of help<br />
codes(amino codes) amino(amino geometry)&lt;/onlyinclude&gt;</pre> <br />
==== Help documents for specific commands ====<br />
<br />
Each command has a short help document describing its usage and options. <br> Syntax:<br> help {topic} {subtopic} <br />
<br />
Example: <onlyinclude>PdbStat> help read<br />
<br />
Read<br />
Syntax: rea[d] {<arg1>} {<arg2>} {<arg3>}<br />
<br />
arg1 can be: coo[rdinates] or con[straints] or seq[uence]<br />
arg2 can be: pdb or con[gen] or cha[rmm] or dis[man]<br />
arg3 can be: the name of the file.<br />
<br />
Examples: read coord pdb filename.pdb<br />
read seq filename<br />
read cons congen filename<br />
read cons diana filename<br />
<br />
Read coordinates/constraints/sequence in specified format from file. This <br />
command is able to open the file read it locating the number of structures <br />
stored and then read them.<br />
<br />
The user needs to type only <read> then a interactive cycle with questions<br />
begins. The program offers the different formats available, so after read<br />
is typed the program offers <coords/cons/seq> and then after that the<br />
program offers the different formats <pdb,disman,....></onlyinclude> <br />
<br />
<br> <br />
<br />
==== Preparation of final CNS coordinates for PDB deposition ====<br />
<br />
Before we deposit our final coordinates we would like to order our models in order of lowest conformation energy. Also, we want to perform backbone superposition of the coordinates using ordered [S(phi) + S(psi) &gt; 1.8] residues only. Finally, we want to rotate the ensemble to a desired orientation, which will be the orientation that appears when a user downloads our coordinates from the Protein Data Bank (www.rcsb.org). The final coordinates are saved in the original (CNS) format and IUPAC format for RPF analysis [2]. <br />
<br />
To prepare the final coordinates file for PDB deposition use the following protocol: <br />
<br />
PdbStat: <br> <onlyinclude><br />
rea coo pdb [filename] #read file with concatenated CNS pdb files<br />
all #select all the models<br />
class #classify the models by energy<br />
order 0.9 #determine ordered residues; phi/psi cut-off 0.9<br />
rmsd best backbone #backbone rmsd<br />
[return] #creates an rmsd output file<br />
write coo pdb [overlayed file] #write overlayed coordinates</onlyinclude> <br />
<br />
Next, open the overlayed coordinates in Molmol and get the desired rotation. Use <tt>writetransform</tt> command to write the rotation matrix to a file. <br> Back in PdbStat: <br> <onlyinclude><br />
rea coo pdb [overlayed.pdb] #read the overlayed coordinates (all models)<br />
rotate file [filename] #apply rotation matrix<br />
write coo pdb [final.pdb] #write new coordinates<br />
to iupac #converts atom nomenclature to IUPAC<br />
write coo pdb [final_iupac.pdb] #write IUPAC coordinates for RPF analysis</onlyinclude> <br />
<br />
==== Selecting specific models / residues / atoms. ====<br />
<br />
A powerful option in PdbStat is the ability to select specific residues and/or atoms for further analysis. This is extremely useful for superposition and RMSD evaluation of selected residues/atoms. <br> Syntax and Examples: <br> <br />
<pre>#syntax<br />
sel[ect] {model(s)} {residue(s)} {atom(s)} <br />
#select backbone atoms of residues 5-25,30-50 in models 1,3-5,7-10<br />
sele 1,3-5,7-10 5-25,30-50 backbone <br />
#select N,C,CA,O atoms of residues 5-50 in all models <br />
sele * 5-50 n,c,ca,o <br />
#combine selection with superposition <br />
sele * 5-50,60-85 *<br />
rmsd sele backbone<br />
</pre> <br />
==== Conversion of coordinate and constraint formats ====<br />
<br />
In the course of a structure refinement we regularly have to convert between different coordinate and constraint formats for different structure programs (i.e., CYANA, XPLOR/CNS, ECEPP). We routinely use PdbStat for this. <br> Examples:<br> <br />
<pre># converting CYANA to XPLOR/CNS coordinates:<br />
rea coor pdb [CYANA.pdb] #read CYANA models (all)<br />
to xplor #converts to xplor; fixes stereospecfic labels <br />
write #answer questions<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns.pdb<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ coor<br />
&gt; WRITER: ... backbone, heavy, full set? (back/heavy/all/select): all<br />
&gt; WRITER: What model&nbsp;?_&nbsp;: all<br />
&gt; WRITER: -- All models to be written <br />
&gt; COORD_writer: Format (pdb/congen/RasMol)&nbsp;?&nbsp;: pdb <br />
# converting CYANA to XPLOR/CNS distance constraints:<br />
rea coor pdb [CYANA.pdb] #read CYANA models; you have to read in a pdb or sequence file before the constraints<br />
rea cons cyana [CYANA.upl] #read CYANA upls file<br />
write #write to CNS format; add 10% to upper bound; make lower bound van der Waals (1.8 A)<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_noe.tbl <br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ cons<br />
&gt; WRITER_constr: Output format <br />
[congen|discover|ecepp|disman|dyana|cyana|diana|xplor|cns]?&nbsp;: cns<br />
&gt; XPLOR_writer: percentage range for upper bound (upp+range)?: 10<br />
&gt; XPLOR_writer: range for lower bound (low-range)?: vdw<br />
&gt; XPLOR_writer: ... writing ... wait <br />
# converting CYANA to XPLOR/CNS dihedral constraints:<br />
rea aco cyana [CYANA.aco]<br />
write<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_dihe.tbl<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ aco <br />
&gt; WRITER_constr: Output format (congen | discover | impact | disman | diana | xplor | cns)?&nbsp;: cns</pre> <br />
==== Constraint Violations ====<br />
<br />
One can analyze constraint violations using PdbStat. It is best to convert the coordinates and constraints to IUPAC format; this is the approach used internally within PSVS. <br> Commands: <onlyinclude><br />
rea coo pdb [filename]<br />
to iupac #convert coordinates to IUPAC format<br />
rea cons [format] [filename] #read distance constraints; specify format<br />
noe to iupac #convert distance constraints to IUPAC<br />
rea aco [format] [filename] #read dihedral constraints; specify format<br />
set cutu 0.2 #set cut-off for distance violations to 0.2 A<br />
see viol noe [sum/ave/center] #see noe violations using sum/average/center averaging<br />
see cutaco 1 #set cut-off for dihedral violations to 1 deg.<br />
see viol aco #see dihedral violations above threshold</onlyinclude> <br />
<br />
==== Sorting of Distance Constraints ====<br />
<br />
There are a number of options in PdbStat for sorting or culling distance constraints. <br> <onlyinclude><br />
cons clean #keep conformationally-restricting constraints; also removes duplicates<br />
noe analysis #NOE statistics (as in PSVS)<br />
noe delete intra #delete all intra NOE constraints<br />
noe keep long #keep only long range (||i-j|| >/= 5) NOE constraints<br />
noe keep ilv #keep NOE constraints consistent with ILV labeling</onlyinclude> <br />
<br />
==== Commands for Obtaining Various Metrics ====<br />
<br />
The “eval”, “show”, and “see” commands allow one to evaluate several types of metrics in a structure or ensemble. <br> <onlyinclude><br />
eval [procheck/rama] #get Procheck/Ramachandran statistics for model(s)<br />
eval dist * 68 sg 119 nd1 #get 68-SG to 119-ND1 distance across all models<br />
eval [phi || psi || omega] 1 #get phi/psi/omega torsion angles in model 1<br />
see lib [residue] #see library definitions for residue type<br />
select 1 119 * #use select and see in tandem<br />
see coo #coordinates for residue 119 in model 1</onlyinclude> <br />
<br />
==== Other Functions ====<br />
<br />
<onlyinclude><br />
# renumbering / resetting residue numbering<br />
reset * 10 #sets first residue in file to 10<br />
reset * #resets coordinates to original<br />
# ordering ensemble using FindCore algorithm [3]:<br />
find [ -bb || -heavy || -all || -noe ]<br />
# contact map generation based on coordinates or constraints<br />
contact<br />
> DRAW_cntct: from coordinates or constraints (coor/cons) ?:_ coor<br />
> MAIN_cntct: What model do you want (1-20) or average ?_ :ave<br />
> do_average_coords(): Making average for backbone atoms <br />
> do_average_coords(): Calculating center of masses <br />
> do_average_coords(): Calling optimal rotation for backbone <br />
> do_average_coords(): Calc. average coordinates backbone <br />
> MAIN_cntct: distance cutoff ?:_ 4.5<br />
> MAIN_cntct: atom type (hydr, heavy, all) ?:_ hydr #writes a postscript file</onlyinclude> <br />
<br />
<br> <br />
<br />
== <span style="font-weight: bold;">Updates</span> ==<br />
<br />
==== Version 5.4<br> ====<br />
<br />
Several new functions have been added to PdbStat and a new version of the program (PdbStat 5.4) was released in April 2011.&nbsp; Below is a summary of new functions added to the program.<br> <br />
<br />
1.&nbsp; Reading and analyzing residual dipolar couplings.<br> <br />
<br />
Example:<br> <br />
<pre># reading two rdc files in cyana format (i.e., after a CYANA&nbsp;3.0 structure calculation with RDCs):<br />
<br />
rea rdc cyana [file1.rdc] #read CYANA rdc file #1<br />
rea rdc cyana [file2.rdc] #read CYANA rdc file #2<br />
set nmedia 2 #set the number of media to 2 <br />
eval dar #get Da and R for each media <br />
#now write the rdc constraints out in CNS format <br />
write dar 1 <br />
write dar 2 <br />
write rdc cns [filename_rdc1.tbl] 1<br />
write rdc cns [filename_rdc2.tbl] 2<br />
</pre> <br />
<br />
2.&nbsp; Reading and converting hydrogen bond constraints.<br> <br />
<br />
Example:<br> <br />
<pre># reading hydrogen bond constraints in CYANA format and converting to CNS:<br />
<br />
rea coo pdb final.pdb #read CYANA&nbsp;pdb file<br />
rea upl cyana hbonds.upl #read CYANA hbonds upl file<br />
rea lol cyana hbonds.lol #read CYANA hbonds lol file <br />
write cons cns [filename_hbond.tbl] #write hbond constraints in CNS format<br />
</pre> <br />
3.&nbsp; Reading and converting metal ion constraints.<br> <br />
<br />
Example:<br> <br />
<pre># reading metal ion constraints in CYANA format and converting to CNS:<br />
<br />
rea coo pdb final.pdb #read CYANA&nbsp;pdb file<br />
rea upl cyana metal.upl #read CYANA metal ion upl file<br />
rea lol cyana metal.lol #read CYANA metal ion lol <br />
write cons cns [filename_metal.tbl] #write metal ion constraints in CNS format<br />
</pre> <br />
4.&nbsp; Converting constraints to Rosetta format<br> <br />
<br />
Example:<br> <br />
<pre># If you have already loaded cons and aco constraints into PdbStat:<br />
<br />
write noe rosetta [outputfile]<br />
write aco rosetta [outputfile]<br />
</pre> <br />
<br />
<br />
<br />
== '''References''' ==<br />
<br />
[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795. ]<br>[http://www.ncbi.nlm.nih.gov/pubmed/15701001?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=4 2. Huang, Y. J., Powers, R., and Montelione, G.T. (2005) Protein NMR Recall, Precision, and F-measure scores (RPF scores): structure quality assessment measures based on information retrieval statistics. ''J. Am. Chem. Soc. 127'', 1665-1674. ]<br>[http://www3.interscience.wiley.com/journal/110445756/abstract 3. Snyder, D. A. and Montelione, G. T. (2005) Clustering algorithms for identifying core atom sets and for assessing the precision of protein structure ensembles. ''Proteins 59'', 673-686. ]<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=PdbStat&diff=3841
PdbStat
2011-10-05T14:20:46Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
The PdbStat program, written and actively developed by our colleague Roberto Tejero (Universita de Valencia), is routinely used in the Montelione lab for the analysis, conversion, and manipulation of coordinate and constraint files for protein structure determination. The program is also an integral component of the Protein Structure Validation Software package [1] used across the NESG consortium. <br />
<br />
The current version of the software is PdbStat 5.1 (July, 2008). There is no separate publication for PdbStat; we cite this program using Bhattacharya et al, 2007 [1]. Also, there is no official manual for the program. Below are a number of basic stand-alone PdbStat commands and common uses. Going forward, we can continue to add to this document as more applications are developed. <br />
<br />
Anyone interested in obtaining the latest version of PdbStat can contact Roberto at: [mailto:roberto.tejero@uv.es roberto.tejero@uv.es] <br> <br />
<br />
== '''Commands and Applications''' ==<br />
<br />
==== Starting the program ====<br />
<br />
<tt>pdbstat</tt><br />
<br />
==== Menu of Commands ====<br />
<br />
<tt>menu</tt> gives a list of commands and keywords recognized by PdbStat (also get this using the command: <tt>help command</tt>) <br />
<br />
Here are the recognized PdbStat commands for version 5.1: <br />
<pre>PdbStat&gt; menu<br />
<br />
Commands/Keywords currently recognized by PDBSTAT<br />
<br />
The commands/keywords currently recognized by PDBSTAT are given below. <br />
Type "help &lt;command&gt;" for more information on each PDBSTAT function.<br />
<br />
align analyze author bye class<br />
clear close chain check chiral<br />
delete debug dump energy evaluate <br />
find fit fix get help<br />
history hydrogen hyper kabsch ident/chain<br />
info initialize log/change load menu<br />
missing order phi quit rama<br />
read relax reset restore rotate<br />
save see set show rmsd<br />
to trans version what write<br />
<br />
homology commands: <br />
get change homology homa<br />
<br />
Other kind/commands/keywords of help<br />
codes(amino codes) amino(amino geometry)&lt;/onlyinclude&gt;</pre> <br />
==== Help documents for specific commands ====<br />
<br />
Each command has a short help document describing its usage and options. <br> Syntax:<br> help {topic} {subtopic} <br />
<br />
Example: <onlyinclude>PdbStat> help read<br />
<br />
Read<br />
Syntax: rea[d] {<arg1>} {<arg2>} {<arg3>}<br />
<br />
arg1 can be: coo[rdinates] or con[straints] or seq[uence]<br />
arg2 can be: pdb or con[gen] or cha[rmm] or dis[man]<br />
arg3 can be: the name of the file.<br />
<br />
Examples: read coord pdb filename.pdb<br />
read seq filename<br />
read cons congen filename<br />
read cons diana filename<br />
<br />
Read coordinates/constraints/sequence in specified format from file. This <br />
command is able to open the file read it locating the number of structures <br />
stored and then read them.<br />
<br />
The user needs to type only <read> then a interactive cycle with questions<br />
begins. The program offers the different formats available, so after read<br />
is typed the program offers <coords/cons/seq> and then after that the<br />
program offers the different formats <pdb,disman,....></onlyinclude> <br />
<br />
<br> <br />
<br />
==== Preparation of final CNS coordinates for PDB deposition ====<br />
<br />
Before we deposit our final coordinates we would like to order our models in order of lowest conformation energy. Also, we want to perform backbone superposition of the coordinates using ordered [S(phi) + S(psi) &gt; 1.8] residues only. Finally, we want to rotate the ensemble to a desired orientation, which will be the orientation that appears when a user downloads our coordinates from the Protein Data Bank (www.rcsb.org). The final coordinates are saved in the original (CNS) format and IUPAC format for RPF analysis [2]. <br />
<br />
To prepare the final coordinates file for PDB deposition use the following protocol: <br />
<br />
PdbStat: <br> <onlyinclude><br />
rea coo pdb [filename] #read file with concatenated CNS pdb files<br />
all #select all the models<br />
class #classify the models by energy<br />
order 0.9 #determine ordered residues; phi/psi cut-off 0.9<br />
rmsd best backbone #backbone rmsd<br />
[return] #creates an rmsd output file<br />
write coo pdb [overlayed file] #write overlayed coordinates</onlyinclude> <br />
<br />
Next, open the overlayed coordinates in Molmol and get the desired rotation. Use <tt>writetransform</tt> command to write the rotation matrix to a file. <br> Back in PdbStat: <br> <onlyinclude><br />
rea coo pdb [overlayed.pdb] #read the overlayed coordinates (all models)<br />
rotate file [filename] #apply rotation matrix<br />
write coo pdb [final.pdb] #write new coordinates<br />
to iupac #converts atom nomenclature to IUPAC<br />
write coo pdb [final_iupac.pdb] #write IUPAC coordinates for RPF analysis</onlyinclude> <br />
<br />
==== Selecting specific models / residues / atoms. ====<br />
<br />
A powerful option in PdbStat is the ability to select specific residues and/or atoms for further analysis. This is extremely useful for superposition and RMSD evaluation of selected residues/atoms. <br> Syntax and Examples: <br> <br />
<pre>#syntax<br />
sel[ect] {model(s)} {residue(s)} {atom(s)} <br />
#select backbone atoms of residues 5-25,30-50 in models 1,3-5,7-10<br />
sele 1,3-5,7-10 5-25,30-50 backbone <br />
#select N,C,CA,O atoms of residues 5-50 in all models <br />
sele * 5-50 n,c,ca,o <br />
#combine selection with superposition <br />
sele * 5-50,60-85 *<br />
rmsd sele backbone<br />
</pre> <br />
==== Conversion of coordinate and constraint formats ====<br />
<br />
In the course of a structure refinement we regularly have to convert between different coordinate and constraint formats for different structure programs (i.e., CYANA, XPLOR/CNS, ECEPP). We routinely use PdbStat for this. <br> Examples:<br> <br />
<pre># converting CYANA to XPLOR/CNS coordinates:<br />
rea coor pdb [CYANA.pdb] #read CYANA models (all)<br />
to xplor #converts to xplor; fixes stereospecfic labels <br />
write #answer questions<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns.pdb<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ coor<br />
&gt; WRITER: ... backbone, heavy, full set? (back/heavy/all/select): all<br />
&gt; WRITER: What model&nbsp;?_&nbsp;: all<br />
&gt; WRITER: -- All models to be written <br />
&gt; COORD_writer: Format (pdb/congen/RasMol)&nbsp;?&nbsp;: pdb <br />
# converting CYANA to XPLOR/CNS distance constraints:<br />
rea coor pdb [CYANA.pdb] #read CYANA models; you have to read in a pdb or sequence file before the constraints<br />
rea cons cyana [CYANA.upl] #read CYANA upls file<br />
write #write to CNS format; add 10% to upper bound; make lower bound van der Waals (1.8 A)<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_noe.tbl <br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ cons<br />
&gt; WRITER_constr: Output format <br />
[congen|discover|ecepp|disman|dyana|cyana|diana|xplor|cns]?&nbsp;: cns<br />
&gt; XPLOR_writer: percentage range for upper bound (upp+range)?: 10<br />
&gt; XPLOR_writer: range for lower bound (low-range)?: vdw<br />
&gt; XPLOR_writer: ... writing ... wait <br />
# converting CYANA to XPLOR/CNS dihedral constraints:<br />
rea aco cyana [CYANA.aco]<br />
write<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_dihe.tbl<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ aco <br />
&gt; WRITER_constr: Output format (congen | discover | impact | disman | diana | xplor | cns)?&nbsp;: cns</pre> <br />
==== Constraint Violations ====<br />
<br />
One can analyze constraint violations using PdbStat. It is best to convert the coordinates and constraints to IUPAC format; this is the approach used internally within PSVS. <br> Commands: <onlyinclude><br />
rea coo pdb [filename]<br />
to iupac #convert coordinates to IUPAC format<br />
rea cons [format] [filename] #read distance constraints; specify format<br />
noe to iupac #convert distance constraints to IUPAC<br />
rea aco [format] [filename] #read dihedral constraints; specify format<br />
set cutu 0.2 #set cut-off for distance violations to 0.2 A<br />
see viol noe [sum/ave/center] #see noe violations using sum/average/center averaging<br />
see cutaco 1 #set cut-off for dihedral violations to 1 deg.<br />
see viol aco #see dihedral violations above threshold</onlyinclude> <br />
<br />
==== Sorting of Distance Constraints ====<br />
<br />
There are a number of options in PdbStat for sorting or culling distance constraints. <br> <onlyinclude><br />
cons clean #keep conformationally-restricting constraints; also removes duplicates<br />
noe analysis #NOE statistics (as in PSVS)<br />
noe delete intra #delete all intra NOE constraints<br />
noe keep long #keep only long range (||i-j|| >/= 5) NOE constraints<br />
noe keep ilv #keep NOE constraints consistent with ILV labeling</onlyinclude> <br />
<br />
==== Commands for Obtaining Various Metrics ====<br />
<br />
The “eval”, “show”, and “see” commands allow one to evaluate several types of metrics in a structure or ensemble. <br> <onlyinclude><br />
eval [procheck/rama] #get Procheck/Ramachandran statistics for model(s)<br />
eval dist * 68 sg 119 nd1 #get 68-SG to 119-ND1 distance across all models<br />
eval [phi || psi || omega] 1 #get phi/psi/omega torsion angles in model 1<br />
see lib [residue] #see library definitions for residue type<br />
select 1 119 * #use select and see in tandem<br />
see coo #coordinates for residue 119 in model 1</onlyinclude> <br />
<br />
==== Other Functions ====<br />
<br />
<onlyinclude><br />
# renumbering / resetting residue numbering<br />
reset * 10 #sets first residue in file to 10<br />
reset * #resets coordinates to original<br />
# ordering ensemble using FindCore algorithm [3]:<br />
find [ -bb || -heavy || -all || -noe ]<br />
# contact map generation based on coordinates or constraints<br />
contact<br />
> DRAW_cntct: from coordinates or constraints (coor/cons) ?:_ coor<br />
> MAIN_cntct: What model do you want (1-20) or average ?_ :ave<br />
> do_average_coords(): Making average for backbone atoms <br />
> do_average_coords(): Calculating center of masses <br />
> do_average_coords(): Calling optimal rotation for backbone <br />
> do_average_coords(): Calc. average coordinates backbone <br />
> MAIN_cntct: distance cutoff ?:_ 4.5<br />
> MAIN_cntct: atom type (hydr, heavy, all) ?:_ hydr #writes a postscript file</onlyinclude> <br />
<br />
<br> <br />
<br />
== <span style="font-weight: bold;">Updates</span> ==<br />
<br />
==== Version 5.4<br> ====<br />
<br />
Several new functions have been added to PdbStat and a new version of the program (PdbStat 5.4) was released in April 2011.&nbsp; Below is a summary of new functions added to the program.<br> <br />
<br />
1.&nbsp; Reading and analyzing residual dipolar couplings.<br> <br />
<br />
Example:<br> <br />
<pre># reading two rdc files in cyana format (i.e., after a CYANA&nbsp;3.0 structure calculation with RDCs):<br />
<br />
rea rdc cyana [file1.rdc] #read CYANA rdc file #1<br />
rea rdc cyana [file2.rdc] #read CYANA rdc file #2<br />
set nmedia 2 #set the number of media to 2 <br />
eval dar #get Da and R for each media <br />
#now write the rdc constraints out in CNS format <br />
write dar 1 <br />
write dar 2 <br />
write rdc cns [filename_rdc1.tbl] 1<br />
write rdc cns [filename_rdc2.tbl] 2<br />
</pre> <br />
==== ====<br />
<br />
2.&nbsp; Reading and converting hydrogen bond constraints.<br> <br />
<br />
Example:<br> <br />
<pre># reading hydrogen bond constraints in CYANA format and converting to CNS:<br />
<br />
rea coo pdb final.pdb #read CYANA&nbsp;pdb file<br />
rea upl cyana hbonds.upl #read CYANA hbonds upl file<br />
rea lol cyana hbonds.lol #read CYANA hbonds lol file <br />
write cons cns [filename_hbond.tbl] #write hbond constraints in CNS format<br />
</pre> <br />
3.&nbsp; Reading and converting metal ion constraints.<br> <br />
<br />
Example:<br> <br />
<pre># reading metal ion constraints in CYANA format and converting to CNS:<br />
<br />
rea coo pdb final.pdb #read CYANA&nbsp;pdb file<br />
rea upl cyana metal.upl #read CYANA metal ion upl file<br />
rea lol cyana metal.lol #read CYANA metal ion lol <br />
write cons cns [filename_metal.tbl] #write metal ion constraints in CNS format<br />
</pre> <br />
==== ====<br />
<br />
==== &nbsp;&nbsp;&nbsp; ====<br />
<br />
== '''References''' ==<br />
<br />
[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795. ]<br>[http://www.ncbi.nlm.nih.gov/pubmed/15701001?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=4 2. Huang, Y. J., Powers, R., and Montelione, G.T. (2005) Protein NMR Recall, Precision, and F-measure scores (RPF scores): structure quality assessment measures based on information retrieval statistics. ''J. Am. Chem. Soc. 127'', 1665-1674. ]<br>[http://www3.interscience.wiley.com/journal/110445756/abstract 3. Snyder, D. A. and Montelione, G. T. (2005) Clustering algorithms for identifying core atom sets and for assessing the precision of protein structure ensembles. ''Proteins 59'', 673-686. ]<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=PdbStat&diff=3840
PdbStat
2011-10-05T14:18:11Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
The PdbStat program, written and actively developed by our colleague Roberto Tejero (Universita de Valencia), is routinely used in the Montelione lab for the analysis, conversion, and manipulation of coordinate and constraint files for protein structure determination. The program is also an integral component of the Protein Structure Validation Software package [1] used across the NESG consortium. <br />
<br />
The current version of the software is PdbStat 5.1 (July, 2008). There is no separate publication for PdbStat; we cite this program using Bhattacharya et al, 2007 [1]. Also, there is no official manual for the program. Below are a number of basic stand-alone PdbStat commands and common uses. Going forward, we can continue to add to this document as more applications are developed. <br />
<br />
Anyone interested in obtaining the latest version of PdbStat can contact Roberto at: [mailto:roberto.tejero@uv.es roberto.tejero@uv.es] <br> <br />
<br />
== '''Commands and Applications''' ==<br />
<br />
==== Starting the program ====<br />
<br />
<tt>pdbstat</tt><br />
<br />
==== Menu of Commands ====<br />
<br />
<tt>menu</tt> gives a list of commands and keywords recognized by PdbStat (also get this using the command: <tt>help command</tt>) <br />
<br />
Here are the recognized PdbStat commands for version 5.1: <br />
<pre>PdbStat&gt; menu<br />
<br />
Commands/Keywords currently recognized by PDBSTAT<br />
<br />
The commands/keywords currently recognized by PDBSTAT are given below. <br />
Type "help &lt;command&gt;" for more information on each PDBSTAT function.<br />
<br />
align analyze author bye class<br />
clear close chain check chiral<br />
delete debug dump energy evaluate <br />
find fit fix get help<br />
history hydrogen hyper kabsch ident/chain<br />
info initialize log/change load menu<br />
missing order phi quit rama<br />
read relax reset restore rotate<br />
save see set show rmsd<br />
to trans version what write<br />
<br />
homology commands: <br />
get change homology homa<br />
<br />
Other kind/commands/keywords of help<br />
codes(amino codes) amino(amino geometry)&lt;/onlyinclude&gt;</pre> <br />
==== Help documents for specific commands ====<br />
<br />
Each command has a short help document describing its usage and options. <br> Syntax:<br> help {topic} {subtopic} <br />
<br />
Example: <onlyinclude>PdbStat> help read<br />
<br />
Read<br />
Syntax: rea[d] {<arg1>} {<arg2>} {<arg3>}<br />
<br />
arg1 can be: coo[rdinates] or con[straints] or seq[uence]<br />
arg2 can be: pdb or con[gen] or cha[rmm] or dis[man]<br />
arg3 can be: the name of the file.<br />
<br />
Examples: read coord pdb filename.pdb<br />
read seq filename<br />
read cons congen filename<br />
read cons diana filename<br />
<br />
Read coordinates/constraints/sequence in specified format from file. This <br />
command is able to open the file read it locating the number of structures <br />
stored and then read them.<br />
<br />
The user needs to type only <read> then a interactive cycle with questions<br />
begins. The program offers the different formats available, so after read<br />
is typed the program offers <coords/cons/seq> and then after that the<br />
program offers the different formats <pdb,disman,....></onlyinclude> <br />
<br />
<br> <br />
<br />
==== Preparation of final CNS coordinates for PDB deposition ====<br />
<br />
Before we deposit our final coordinates we would like to order our models in order of lowest conformation energy. Also, we want to perform backbone superposition of the coordinates using ordered [S(phi) + S(psi) &gt; 1.8] residues only. Finally, we want to rotate the ensemble to a desired orientation, which will be the orientation that appears when a user downloads our coordinates from the Protein Data Bank (www.rcsb.org). The final coordinates are saved in the original (CNS) format and IUPAC format for RPF analysis [2]. <br />
<br />
To prepare the final coordinates file for PDB deposition use the following protocol: <br />
<br />
PdbStat: <br> <onlyinclude><br />
rea coo pdb [filename] #read file with concatenated CNS pdb files<br />
all #select all the models<br />
class #classify the models by energy<br />
order 0.9 #determine ordered residues; phi/psi cut-off 0.9<br />
rmsd best backbone #backbone rmsd<br />
[return] #creates an rmsd output file<br />
write coo pdb [overlayed file] #write overlayed coordinates</onlyinclude> <br />
<br />
Next, open the overlayed coordinates in Molmol and get the desired rotation. Use <tt>writetransform</tt> command to write the rotation matrix to a file. <br> Back in PdbStat: <br> <onlyinclude><br />
rea coo pdb [overlayed.pdb] #read the overlayed coordinates (all models)<br />
rotate file [filename] #apply rotation matrix<br />
write coo pdb [final.pdb] #write new coordinates<br />
to iupac #converts atom nomenclature to IUPAC<br />
write coo pdb [final_iupac.pdb] #write IUPAC coordinates for RPF analysis</onlyinclude> <br />
<br />
==== Selecting specific models / residues / atoms. ====<br />
<br />
A powerful option in PdbStat is the ability to select specific residues and/or atoms for further analysis. This is extremely useful for superposition and RMSD evaluation of selected residues/atoms. <br> Syntax and Examples: <br> <br />
<pre>#syntax<br />
sel[ect] {model(s)} {residue(s)} {atom(s)} <br />
#select backbone atoms of residues 5-25,30-50 in models 1,3-5,7-10<br />
sele 1,3-5,7-10 5-25,30-50 backbone <br />
#select N,C,CA,O atoms of residues 5-50 in all models <br />
sele * 5-50 n,c,ca,o <br />
#combine selection with superposition <br />
sele * 5-50,60-85 *<br />
rmsd sele backbone<br />
</pre> <br />
==== Conversion of coordinate and constraint formats ====<br />
<br />
In the course of a structure refinement we regularly have to convert between different coordinate and constraint formats for different structure programs (i.e., CYANA, XPLOR/CNS, ECEPP). We routinely use PdbStat for this. <br> Examples:<br> <br />
<pre># converting CYANA to XPLOR/CNS coordinates:<br />
rea coor pdb [CYANA.pdb] #read CYANA models (all)<br />
to xplor #converts to xplor; fixes stereospecfic labels <br />
write #answer questions<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns.pdb<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ coor<br />
&gt; WRITER: ... backbone, heavy, full set? (back/heavy/all/select): all<br />
&gt; WRITER: What model&nbsp;?_&nbsp;: all<br />
&gt; WRITER: -- All models to be written <br />
&gt; COORD_writer: Format (pdb/congen/RasMol)&nbsp;?&nbsp;: pdb <br />
# converting CYANA to XPLOR/CNS distance constraints:<br />
rea coor pdb [CYANA.pdb] #read CYANA models; you have to read in a pdb or sequence file before the constraints<br />
rea cons cyana [CYANA.upl] #read CYANA upls file<br />
write #write to CNS format; add 10% to upper bound; make lower bound van der Waals (1.8 A)<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_noe.tbl <br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ cons<br />
&gt; WRITER_constr: Output format <br />
[congen|discover|ecepp|disman|dyana|cyana|diana|xplor|cns]?&nbsp;: cns<br />
&gt; XPLOR_writer: percentage range for upper bound (upp+range)?: 10<br />
&gt; XPLOR_writer: range for lower bound (low-range)?: vdw<br />
&gt; XPLOR_writer: ... writing ... wait <br />
# converting CYANA to XPLOR/CNS dihedral constraints:<br />
rea aco cyana [CYANA.aco]<br />
write<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_dihe.tbl<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ aco <br />
&gt; WRITER_constr: Output format (congen | discover | impact | disman | diana | xplor | cns)?&nbsp;: cns</pre> <br />
==== Constraint Violations ====<br />
<br />
One can analyze constraint violations using PdbStat. It is best to convert the coordinates and constraints to IUPAC format; this is the approach used internally within PSVS. <br> Commands: <onlyinclude><br />
rea coo pdb [filename]<br />
to iupac #convert coordinates to IUPAC format<br />
rea cons [format] [filename] #read distance constraints; specify format<br />
noe to iupac #convert distance constraints to IUPAC<br />
rea aco [format] [filename] #read dihedral constraints; specify format<br />
set cutu 0.2 #set cut-off for distance violations to 0.2 A<br />
see viol noe [sum/ave/center] #see noe violations using sum/average/center averaging<br />
see cutaco 1 #set cut-off for dihedral violations to 1 deg.<br />
see viol aco #see dihedral violations above threshold</onlyinclude> <br />
<br />
==== Sorting of Distance Constraints ====<br />
<br />
There are a number of options in PdbStat for sorting or culling distance constraints. <br> <onlyinclude><br />
cons clean #keep conformationally-restricting constraints; also removes duplicates<br />
noe analysis #NOE statistics (as in PSVS)<br />
noe delete intra #delete all intra NOE constraints<br />
noe keep long #keep only long range (||i-j|| >/= 5) NOE constraints<br />
noe keep ilv #keep NOE constraints consistent with ILV labeling</onlyinclude> <br />
<br />
==== Commands for Obtaining Various Metrics ====<br />
<br />
The “eval”, “show”, and “see” commands allow one to evaluate several types of metrics in a structure or ensemble. <br> <onlyinclude><br />
eval [procheck/rama] #get Procheck/Ramachandran statistics for model(s)<br />
eval dist * 68 sg 119 nd1 #get 68-SG to 119-ND1 distance across all models<br />
eval [phi || psi || omega] 1 #get phi/psi/omega torsion angles in model 1<br />
see lib [residue] #see library definitions for residue type<br />
select 1 119 * #use select and see in tandem<br />
see coo #coordinates for residue 119 in model 1</onlyinclude> <br />
<br />
==== Other Functions ====<br />
<br />
<onlyinclude><br />
# renumbering / resetting residue numbering<br />
reset * 10 #sets first residue in file to 10<br />
reset * #resets coordinates to original<br />
# ordering ensemble using FindCore algorithm [3]:<br />
find [ -bb || -heavy || -all || -noe ]<br />
# contact map generation based on coordinates or constraints<br />
contact<br />
> DRAW_cntct: from coordinates or constraints (coor/cons) ?:_ coor<br />
> MAIN_cntct: What model do you want (1-20) or average ?_ :ave<br />
> do_average_coords(): Making average for backbone atoms <br />
> do_average_coords(): Calculating center of masses <br />
> do_average_coords(): Calling optimal rotation for backbone <br />
> do_average_coords(): Calc. average coordinates backbone <br />
> MAIN_cntct: distance cutoff ?:_ 4.5<br />
> MAIN_cntct: atom type (hydr, heavy, all) ?:_ hydr #writes a postscript file</onlyinclude> <br />
<br />
<br> <br />
<br />
== <span style="font-weight: bold;">Updates</span> ==<br />
<br />
==== Version 5.4<br> ====<br />
<br />
Several new functions have been added to PdbStat and a new version of the program (PdbStat 5.4) was released in April 2011.&nbsp; Below is a summary of new functions added to the program.<br> <br />
<br />
1.&nbsp; Reading and analyzing residual dipolar couplings.<br> <br />
<br />
Example:<br> <br />
<pre># reading two rdc files in cyana format (i.e., after a CYANA&nbsp;3.0 structure calculation with RDCs):<br />
<br />
rea rdc cyana [file1.rdc] #read CYANA rdc file #1<br />
rea rdc cyana [file2.rdc] #read CYANA rdc file #2<br />
set nmedia 2 #set the number of media to 2 <br />
eval dar #get Da and R for each media <br />
#now write the rdc constraints out in CNS format <br />
write dar 1 <br />
write dar 2 <br />
write rdc cns [filename_rdc1.tbl] 1<br />
write rdc cns [filename_rdc2.tbl] 2<br />
</pre> <br />
==== ====<br />
<br />
2.&nbsp; Reading and converting hydrogen bond constraints.<br> <br />
<br />
Example:<br> <br />
<pre># reading hydrogen bond constraints in CYANA format and converting to CNS:<br />
<br />
rea coo pdb final.pdb #read CYANA&nbsp;pdb file<br />
rea upl cyana hbonds.upl #read CYANA hbonds.upl file<br />
rea lol cyana hbonds.lol #read CYANA hbonds.lol <br />
write cons cns [filename_hbond.tbl] #write hbond constraints in CNS format<br />
</pre> <br />
==== &nbsp;&nbsp;&nbsp; ====<br />
<br />
== '''References''' ==<br />
<br />
[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795. ]<br>[http://www.ncbi.nlm.nih.gov/pubmed/15701001?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=4 2. Huang, Y. J., Powers, R., and Montelione, G.T. (2005) Protein NMR Recall, Precision, and F-measure scores (RPF scores): structure quality assessment measures based on information retrieval statistics. ''J. Am. Chem. Soc. 127'', 1665-1674. ]<br>[http://www3.interscience.wiley.com/journal/110445756/abstract 3. Snyder, D. A. and Montelione, G. T. (2005) Clustering algorithms for identifying core atom sets and for assessing the precision of protein structure ensembles. ''Proteins 59'', 673-686. ]<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=PdbStat&diff=3839
PdbStat
2011-10-05T14:16:23Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
The PdbStat program, written and actively developed by our colleague Roberto Tejero (Universita de Valencia), is routinely used in the Montelione lab for the analysis, conversion, and manipulation of coordinate and constraint files for protein structure determination. The program is also an integral component of the Protein Structure Validation Software package [1] used across the NESG consortium. <br />
<br />
The current version of the software is PdbStat 5.1 (July, 2008). There is no separate publication for PdbStat; we cite this program using Bhattacharya et al, 2007 [1]. Also, there is no official manual for the program. Below are a number of basic stand-alone PdbStat commands and common uses. Going forward, we can continue to add to this document as more applications are developed. <br />
<br />
Anyone interested in obtaining the latest version of PdbStat can contact Roberto at: [mailto:roberto.tejero@uv.es roberto.tejero@uv.es] <br> <br />
<br />
== '''Commands and Applications''' ==<br />
<br />
==== Starting the program ====<br />
<br />
<tt>pdbstat</tt><br />
<br />
==== Menu of Commands ====<br />
<br />
<tt>menu</tt> gives a list of commands and keywords recognized by PdbStat (also get this using the command: <tt>help command</tt>) <br />
<br />
Here are the recognized PdbStat commands for version 5.1: <br />
<pre>PdbStat&gt; menu<br />
<br />
Commands/Keywords currently recognized by PDBSTAT<br />
<br />
The commands/keywords currently recognized by PDBSTAT are given below. <br />
Type "help &lt;command&gt;" for more information on each PDBSTAT function.<br />
<br />
align analyze author bye class<br />
clear close chain check chiral<br />
delete debug dump energy evaluate <br />
find fit fix get help<br />
history hydrogen hyper kabsch ident/chain<br />
info initialize log/change load menu<br />
missing order phi quit rama<br />
read relax reset restore rotate<br />
save see set show rmsd<br />
to trans version what write<br />
<br />
homology commands: <br />
get change homology homa<br />
<br />
Other kind/commands/keywords of help<br />
codes(amino codes) amino(amino geometry)&lt;/onlyinclude&gt;</pre> <br />
==== Help documents for specific commands ====<br />
<br />
Each command has a short help document describing its usage and options. <br> Syntax:<br> help {topic} {subtopic} <br />
<br />
Example: <onlyinclude>PdbStat> help read<br />
<br />
Read<br />
Syntax: rea[d] {<arg1>} {<arg2>} {<arg3>}<br />
<br />
arg1 can be: coo[rdinates] or con[straints] or seq[uence]<br />
arg2 can be: pdb or con[gen] or cha[rmm] or dis[man]<br />
arg3 can be: the name of the file.<br />
<br />
Examples: read coord pdb filename.pdb<br />
read seq filename<br />
read cons congen filename<br />
read cons diana filename<br />
<br />
Read coordinates/constraints/sequence in specified format from file. This <br />
command is able to open the file read it locating the number of structures <br />
stored and then read them.<br />
<br />
The user needs to type only <read> then a interactive cycle with questions<br />
begins. The program offers the different formats available, so after read<br />
is typed the program offers <coords/cons/seq> and then after that the<br />
program offers the different formats <pdb,disman,....></onlyinclude> <br />
<br />
<br> <br />
<br />
==== Preparation of final CNS coordinates for PDB deposition ====<br />
<br />
Before we deposit our final coordinates we would like to order our models in order of lowest conformation energy. Also, we want to perform backbone superposition of the coordinates using ordered [S(phi) + S(psi) &gt; 1.8] residues only. Finally, we want to rotate the ensemble to a desired orientation, which will be the orientation that appears when a user downloads our coordinates from the Protein Data Bank (www.rcsb.org). The final coordinates are saved in the original (CNS) format and IUPAC format for RPF analysis [2]. <br />
<br />
To prepare the final coordinates file for PDB deposition use the following protocol: <br />
<br />
PdbStat: <br> <onlyinclude><br />
rea coo pdb [filename] #read file with concatenated CNS pdb files<br />
all #select all the models<br />
class #classify the models by energy<br />
order 0.9 #determine ordered residues; phi/psi cut-off 0.9<br />
rmsd best backbone #backbone rmsd<br />
[return] #creates an rmsd output file<br />
write coo pdb [overlayed file] #write overlayed coordinates</onlyinclude> <br />
<br />
Next, open the overlayed coordinates in Molmol and get the desired rotation. Use <tt>writetransform</tt> command to write the rotation matrix to a file. <br> Back in PdbStat: <br> <onlyinclude><br />
rea coo pdb [overlayed.pdb] #read the overlayed coordinates (all models)<br />
rotate file [filename] #apply rotation matrix<br />
write coo pdb [final.pdb] #write new coordinates<br />
to iupac #converts atom nomenclature to IUPAC<br />
write coo pdb [final_iupac.pdb] #write IUPAC coordinates for RPF analysis</onlyinclude> <br />
<br />
==== Selecting specific models / residues / atoms. ====<br />
<br />
A powerful option in PdbStat is the ability to select specific residues and/or atoms for further analysis. This is extremely useful for superposition and RMSD evaluation of selected residues/atoms. <br> Syntax and Examples: <br> <br />
<pre>#syntax<br />
sel[ect] {model(s)} {residue(s)} {atom(s)} <br />
#select backbone atoms of residues 5-25,30-50 in models 1,3-5,7-10<br />
sele 1,3-5,7-10 5-25,30-50 backbone <br />
#select N,C,CA,O atoms of residues 5-50 in all models <br />
sele * 5-50 n,c,ca,o <br />
#combine selection with superposition <br />
sele * 5-50,60-85 *<br />
rmsd sele backbone<br />
</pre> <br />
==== Conversion of coordinate and constraint formats ====<br />
<br />
In the course of a structure refinement we regularly have to convert between different coordinate and constraint formats for different structure programs (i.e., CYANA, XPLOR/CNS, ECEPP). We routinely use PdbStat for this. <br> Examples:<br> <br />
<pre># converting CYANA to XPLOR/CNS coordinates:<br />
rea coor pdb [CYANA.pdb] #read CYANA models (all)<br />
to xplor #converts to xplor; fixes stereospecfic labels <br />
write #answer questions<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns.pdb<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ coor<br />
&gt; WRITER: ... backbone, heavy, full set? (back/heavy/all/select): all<br />
&gt; WRITER: What model&nbsp;?_&nbsp;: all<br />
&gt; WRITER: -- All models to be written <br />
&gt; COORD_writer: Format (pdb/congen/RasMol)&nbsp;?&nbsp;: pdb <br />
# converting CYANA to XPLOR/CNS distance constraints:<br />
rea coor pdb [CYANA.pdb] #read CYANA models; you have to read in a pdb or sequence file before the constraints<br />
rea cons cyana [CYANA.upl] #read CYANA upls file<br />
write #write to CNS format; add 10% to upper bound; make lower bound van der Waals (1.8 A)<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_noe.tbl <br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ cons<br />
&gt; WRITER_constr: Output format <br />
[congen|discover|ecepp|disman|dyana|cyana|diana|xplor|cns]?&nbsp;: cns<br />
&gt; XPLOR_writer: percentage range for upper bound (upp+range)?: 10<br />
&gt; XPLOR_writer: range for lower bound (low-range)?: vdw<br />
&gt; XPLOR_writer: ... writing ... wait <br />
# converting CYANA to XPLOR/CNS dihedral constraints:<br />
rea aco cyana [CYANA.aco]<br />
write<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_dihe.tbl<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ aco <br />
&gt; WRITER_constr: Output format (congen | discover | impact | disman | diana | xplor | cns)?&nbsp;: cns</pre> <br />
==== Constraint Violations ====<br />
<br />
One can analyze constraint violations using PdbStat. It is best to convert the coordinates and constraints to IUPAC format; this is the approach used internally within PSVS. <br> Commands: <onlyinclude><br />
rea coo pdb [filename]<br />
to iupac #convert coordinates to IUPAC format<br />
rea cons [format] [filename] #read distance constraints; specify format<br />
noe to iupac #convert distance constraints to IUPAC<br />
rea aco [format] [filename] #read dihedral constraints; specify format<br />
set cutu 0.2 #set cut-off for distance violations to 0.2 A<br />
see viol noe [sum/ave/center] #see noe violations using sum/average/center averaging<br />
see cutaco 1 #set cut-off for dihedral violations to 1 deg.<br />
see viol aco #see dihedral violations above threshold</onlyinclude> <br />
<br />
==== Sorting of Distance Constraints ====<br />
<br />
There are a number of options in PdbStat for sorting or culling distance constraints. <br> <onlyinclude><br />
cons clean #keep conformationally-restricting constraints; also removes duplicates<br />
noe analysis #NOE statistics (as in PSVS)<br />
noe delete intra #delete all intra NOE constraints<br />
noe keep long #keep only long range (||i-j|| >/= 5) NOE constraints<br />
noe keep ilv #keep NOE constraints consistent with ILV labeling</onlyinclude> <br />
<br />
==== Commands for Obtaining Various Metrics ====<br />
<br />
The “eval”, “show”, and “see” commands allow one to evaluate several types of metrics in a structure or ensemble. <br> <onlyinclude><br />
eval [procheck/rama] #get Procheck/Ramachandran statistics for model(s)<br />
eval dist * 68 sg 119 nd1 #get 68-SG to 119-ND1 distance across all models<br />
eval [phi || psi || omega] 1 #get phi/psi/omega torsion angles in model 1<br />
see lib [residue] #see library definitions for residue type<br />
select 1 119 * #use select and see in tandem<br />
see coo #coordinates for residue 119 in model 1</onlyinclude> <br />
<br />
==== Other Functions ====<br />
<br />
<onlyinclude><br />
# renumbering / resetting residue numbering<br />
reset * 10 #sets first residue in file to 10<br />
reset * #resets coordinates to original<br />
# ordering ensemble using FindCore algorithm [3]:<br />
find [ -bb || -heavy || -all || -noe ]<br />
# contact map generation based on coordinates or constraints<br />
contact<br />
> DRAW_cntct: from coordinates or constraints (coor/cons) ?:_ coor<br />
> MAIN_cntct: What model do you want (1-20) or average ?_ :ave<br />
> do_average_coords(): Making average for backbone atoms <br />
> do_average_coords(): Calculating center of masses <br />
> do_average_coords(): Calling optimal rotation for backbone <br />
> do_average_coords(): Calc. average coordinates backbone <br />
> MAIN_cntct: distance cutoff ?:_ 4.5<br />
> MAIN_cntct: atom type (hydr, heavy, all) ?:_ hydr #writes a postscript file</onlyinclude> <br />
<br />
<br> <br />
<br />
== <span style="font-weight: bold;">Updates</span> ==<br />
<br />
==== Version 5.4<br> ====<br />
<br />
Several new functions have been added to PdbStat and a new version of the program (PdbStat 5.4) was released in April 2011.&nbsp; Below is a summary of new functions added to the program.<br> <br />
<br />
1.&nbsp; Reading and analyzing residual dipolar couplings.<br> <br />
<br />
Example:<br> <br />
<pre># reading two rdc files in cyana format (i.e., after a CYANA&nbsp;3.0 structure calculation with RDCs):<br />
<br />
rea rdc cyana [file1.rdc] #read CYANA rdc file #1<br />
rea rdc cyana [file2.rdc] #read CYANA rdc file #2<br />
set nmedia 2 #set the number of media to 2 <br />
eval dar #get Da and R for each media <br />
#now write the rdc constraints out in CNS format <br />
write dar 1 <br />
write dar 2 <br />
write rdc cns [filename_rdc1.tbl] 1<br />
write rdc cns [filename_rdc2.tbl] 2<br />
<br />
<br />
</pre> <br />
==== ====<br />
<br />
2.&nbsp; Reading and converting hydrogen bond constraints.<br> <br />
<br />
Example:<br> <br />
<pre># reading hydrogen bond constraints in CYANA format and converting to CNS:<br />
<br />
rea coo pdb final.pdb #read CYANA&nbsp;pdb file<br />
rea upl cyana hbonds.upl #read CYANA hbonds.upl file<br />
rea lol cyana hbonds.lol #read CYANA hbonds.lol <br />
write cons cns [filename_hbond.tbl] #write hbond constraints in CNS format<br />
<br />
<br />
</pre> <br />
==== ====<br />
<br />
==== &nbsp;&nbsp;&nbsp; ====<br />
<br />
== '''References''' ==<br />
<br />
[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795. ]<br>[http://www.ncbi.nlm.nih.gov/pubmed/15701001?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=4 2. Huang, Y. J., Powers, R., and Montelione, G.T. (2005) Protein NMR Recall, Precision, and F-measure scores (RPF scores): structure quality assessment measures based on information retrieval statistics. ''J. Am. Chem. Soc. 127'', 1665-1674. ]<br>[http://www3.interscience.wiley.com/journal/110445756/abstract 3. Snyder, D. A. and Montelione, G. T. (2005) Clustering algorithms for identifying core atom sets and for assessing the precision of protein structure ensembles. ''Proteins 59'', 673-686. ]<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=PdbStat&diff=3838
PdbStat
2011-10-05T14:11:18Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
The PdbStat program, written and actively developed by our colleague Roberto Tejero (Universita de Valencia), is routinely used in the Montelione lab for the analysis, conversion, and manipulation of coordinate and constraint files for protein structure determination. The program is also an integral component of the Protein Structure Validation Software package [1] used across the NESG consortium. <br />
<br />
The current version of the software is PdbStat 5.1 (July, 2008). There is no separate publication for PdbStat; we cite this program using Bhattacharya et al, 2007 [1]. Also, there is no official manual for the program. Below are a number of basic stand-alone PdbStat commands and common uses. Going forward, we can continue to add to this document as more applications are developed. <br />
<br />
Anyone interested in obtaining the latest version of PdbStat can contact Roberto at: [mailto:roberto.tejero@uv.es roberto.tejero@uv.es] <br> <br />
<br />
== '''Commands and Applications''' ==<br />
<br />
==== Starting the program ====<br />
<br />
<tt>pdbstat</tt><br />
<br />
==== Menu of Commands ====<br />
<br />
<tt>menu</tt> gives a list of commands and keywords recognized by PdbStat (also get this using the command: <tt>help command</tt>) <br />
<br />
Here are the recognized PdbStat commands for version 5.1: <br />
<pre>PdbStat&gt; menu<br />
<br />
Commands/Keywords currently recognized by PDBSTAT<br />
<br />
The commands/keywords currently recognized by PDBSTAT are given below. <br />
Type "help &lt;command&gt;" for more information on each PDBSTAT function.<br />
<br />
align analyze author bye class<br />
clear close chain check chiral<br />
delete debug dump energy evaluate <br />
find fit fix get help<br />
history hydrogen hyper kabsch ident/chain<br />
info initialize log/change load menu<br />
missing order phi quit rama<br />
read relax reset restore rotate<br />
save see set show rmsd<br />
to trans version what write<br />
<br />
homology commands: <br />
get change homology homa<br />
<br />
Other kind/commands/keywords of help<br />
codes(amino codes) amino(amino geometry)&lt;/onlyinclude&gt;</pre> <br />
==== Help documents for specific commands ====<br />
<br />
Each command has a short help document describing its usage and options. <br> Syntax:<br> help {topic} {subtopic} <br />
<br />
Example: <onlyinclude>PdbStat> help read<br />
<br />
Read<br />
Syntax: rea[d] {<arg1>} {<arg2>} {<arg3>}<br />
<br />
arg1 can be: coo[rdinates] or con[straints] or seq[uence]<br />
arg2 can be: pdb or con[gen] or cha[rmm] or dis[man]<br />
arg3 can be: the name of the file.<br />
<br />
Examples: read coord pdb filename.pdb<br />
read seq filename<br />
read cons congen filename<br />
read cons diana filename<br />
<br />
Read coordinates/constraints/sequence in specified format from file. This <br />
command is able to open the file read it locating the number of structures <br />
stored and then read them.<br />
<br />
The user needs to type only <read> then a interactive cycle with questions<br />
begins. The program offers the different formats available, so after read<br />
is typed the program offers <coords/cons/seq> and then after that the<br />
program offers the different formats <pdb,disman,....></onlyinclude> <br />
<br />
<br> <br />
<br />
==== Preparation of final CNS coordinates for PDB deposition ====<br />
<br />
Before we deposit our final coordinates we would like to order our models in order of lowest conformation energy. Also, we want to perform backbone superposition of the coordinates using ordered [S(phi) + S(psi) &gt; 1.8] residues only. Finally, we want to rotate the ensemble to a desired orientation, which will be the orientation that appears when a user downloads our coordinates from the Protein Data Bank (www.rcsb.org). The final coordinates are saved in the original (CNS) format and IUPAC format for RPF analysis [2]. <br />
<br />
To prepare the final coordinates file for PDB deposition use the following protocol: <br />
<br />
PdbStat: <br> <onlyinclude><br />
rea coo pdb [filename] #read file with concatenated CNS pdb files<br />
all #select all the models<br />
class #classify the models by energy<br />
order 0.9 #determine ordered residues; phi/psi cut-off 0.9<br />
rmsd best backbone #backbone rmsd<br />
[return] #creates an rmsd output file<br />
write coo pdb [overlayed file] #write overlayed coordinates</onlyinclude> <br />
<br />
Next, open the overlayed coordinates in Molmol and get the desired rotation. Use <tt>writetransform</tt> command to write the rotation matrix to a file. <br> Back in PdbStat: <br> <onlyinclude><br />
rea coo pdb [overlayed.pdb] #read the overlayed coordinates (all models)<br />
rotate file [filename] #apply rotation matrix<br />
write coo pdb [final.pdb] #write new coordinates<br />
to iupac #converts atom nomenclature to IUPAC<br />
write coo pdb [final_iupac.pdb] #write IUPAC coordinates for RPF analysis</onlyinclude> <br />
<br />
==== Selecting specific models / residues / atoms. ====<br />
<br />
A powerful option in PdbStat is the ability to select specific residues and/or atoms for further analysis. This is extremely useful for superposition and RMSD evaluation of selected residues/atoms. <br> Syntax and Examples: <br> <br />
<pre>#syntax<br />
sel[ect] {model(s)} {residue(s)} {atom(s)} <br />
#select backbone atoms of residues 5-25,30-50 in models 1,3-5,7-10<br />
sele 1,3-5,7-10 5-25,30-50 backbone <br />
#select N,C,CA,O atoms of residues 5-50 in all models <br />
sele * 5-50 n,c,ca,o <br />
#combine selection with superposition <br />
sele * 5-50,60-85 *<br />
rmsd sele backbone<br />
</pre> <br />
==== Conversion of coordinate and constraint formats ====<br />
<br />
In the course of a structure refinement we regularly have to convert between different coordinate and constraint formats for different structure programs (i.e., CYANA, XPLOR/CNS, ECEPP). We routinely use PdbStat for this. <br> Examples:<br> <br />
<pre># converting CYANA to XPLOR/CNS coordinates:<br />
rea coor pdb [CYANA.pdb] #read CYANA models (all)<br />
to xplor #converts to xplor; fixes stereospecfic labels <br />
write #answer questions<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns.pdb<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ coor<br />
&gt; WRITER: ... backbone, heavy, full set? (back/heavy/all/select): all<br />
&gt; WRITER: What model&nbsp;?_&nbsp;: all<br />
&gt; WRITER: -- All models to be written <br />
&gt; COORD_writer: Format (pdb/congen/RasMol)&nbsp;?&nbsp;: pdb <br />
# converting CYANA to XPLOR/CNS distance constraints:<br />
rea coor pdb [CYANA.pdb] #read CYANA models; you have to read in a pdb or sequence file before the constraints<br />
rea cons cyana [CYANA.upl] #read CYANA upls file<br />
write #write to CNS format; add 10% to upper bound; make lower bound van der Waals (1.8 A)<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_noe.tbl <br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ cons<br />
&gt; WRITER_constr: Output format <br />
[congen|discover|ecepp|disman|dyana|cyana|diana|xplor|cns]?&nbsp;: cns<br />
&gt; XPLOR_writer: percentage range for upper bound (upp+range)?: 10<br />
&gt; XPLOR_writer: range for lower bound (low-range)?: vdw<br />
&gt; XPLOR_writer: ... writing ... wait <br />
# converting CYANA to XPLOR/CNS dihedral constraints:<br />
rea aco cyana [CYANA.aco]<br />
write<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_dihe.tbl<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ aco <br />
&gt; WRITER_constr: Output format (congen | discover | impact | disman | diana | xplor | cns)?&nbsp;: cns</pre> <br />
==== Constraint Violations ====<br />
<br />
One can analyze constraint violations using PdbStat. It is best to convert the coordinates and constraints to IUPAC format; this is the approach used internally within PSVS. <br> Commands: <onlyinclude><br />
rea coo pdb [filename]<br />
to iupac #convert coordinates to IUPAC format<br />
rea cons [format] [filename] #read distance constraints; specify format<br />
noe to iupac #convert distance constraints to IUPAC<br />
rea aco [format] [filename] #read dihedral constraints; specify format<br />
set cutu 0.2 #set cut-off for distance violations to 0.2 A<br />
see viol noe [sum/ave/center] #see noe violations using sum/average/center averaging<br />
see cutaco 1 #set cut-off for dihedral violations to 1 deg.<br />
see viol aco #see dihedral violations above threshold</onlyinclude> <br />
<br />
==== Sorting of Distance Constraints ====<br />
<br />
There are a number of options in PdbStat for sorting or culling distance constraints. <br> <onlyinclude><br />
cons clean #keep conformationally-restricting constraints; also removes duplicates<br />
noe analysis #NOE statistics (as in PSVS)<br />
noe delete intra #delete all intra NOE constraints<br />
noe keep long #keep only long range (||i-j|| >/= 5) NOE constraints<br />
noe keep ilv #keep NOE constraints consistent with ILV labeling</onlyinclude> <br />
<br />
==== Commands for Obtaining Various Metrics ====<br />
<br />
The “eval”, “show”, and “see” commands allow one to evaluate several types of metrics in a structure or ensemble. <br> <onlyinclude><br />
eval [procheck/rama] #get Procheck/Ramachandran statistics for model(s)<br />
eval dist * 68 sg 119 nd1 #get 68-SG to 119-ND1 distance across all models<br />
eval [phi || psi || omega] 1 #get phi/psi/omega torsion angles in model 1<br />
see lib [residue] #see library definitions for residue type<br />
select 1 119 * #use select and see in tandem<br />
see coo #coordinates for residue 119 in model 1</onlyinclude> <br />
<br />
==== Other Functions ====<br />
<br />
<onlyinclude><br />
# renumbering / resetting residue numbering<br />
reset * 10 #sets first residue in file to 10<br />
reset * #resets coordinates to original<br />
# ordering ensemble using FindCore algorithm [3]:<br />
find [ -bb || -heavy || -all || -noe ]<br />
# contact map generation based on coordinates or constraints<br />
contact<br />
> DRAW_cntct: from coordinates or constraints (coor/cons) ?:_ coor<br />
> MAIN_cntct: What model do you want (1-20) or average ?_ :ave<br />
> do_average_coords(): Making average for backbone atoms <br />
> do_average_coords(): Calculating center of masses <br />
> do_average_coords(): Calling optimal rotation for backbone <br />
> do_average_coords(): Calc. average coordinates backbone <br />
> MAIN_cntct: distance cutoff ?:_ 4.5<br />
> MAIN_cntct: atom type (hydr, heavy, all) ?:_ hydr #writes a postscript file</onlyinclude> <br />
<br />
<br> <br />
<br />
== <span style="font-weight: bold;">Updates</span> ==<br />
<br />
==== Version 5.4<br> ====<br />
<br />
Several new functions have been added to PdbStat and a new version of the program (PdbStat 5.4) was released in April 2011.&nbsp; Below is a summary of new functions added to the program.<br> <br />
<br />
1.&nbsp; Reading and anlyzing residual dipolar couplings.<br><br />
<br />
Example:<br><br />
<br />
<br />
<pre># reading two rdc files in cyana format (i.e., after a CYANA&nbsp;3.0 structure calculation with RDCs):<br />
<br />
rea rdc cyana [file1.rdc] #read CYANA rdc file #1<br />
rea rdc cyana [file2.rdc] #read CYANA rdc file #2<br />
set nmedia 2 #set the number of media to 2 <br />
eval dar #get Da and R for each media <br />
#now write the rdc constraints out in CNS format <br />
write dar 1 <br />
write dar 2 <br />
write rdc cns [filename_rdc1.tbl] 1<br />
write rdc cns [filename_rdc2.tbl] 2<br />
<br />
<br />
</pre> <br />
==== <br> ====<br />
<br />
<br />
<br />
&nbsp;&nbsp;&nbsp; <br><br />
<br />
== '''References''' ==<br />
<br />
[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795. ]<br>[http://www.ncbi.nlm.nih.gov/pubmed/15701001?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=4 2. Huang, Y. J., Powers, R., and Montelione, G.T. (2005) Protein NMR Recall, Precision, and F-measure scores (RPF scores): structure quality assessment measures based on information retrieval statistics. ''J. Am. Chem. Soc. 127'', 1665-1674. ]<br>[http://www3.interscience.wiley.com/journal/110445756/abstract 3. Snyder, D. A. and Montelione, G. T. (2005) Clustering algorithms for identifying core atom sets and for assessing the precision of protein structure ensembles. ''Proteins 59'', 673-686. ]<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=PdbStat&diff=3837
PdbStat
2011-10-05T13:56:09Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
The PdbStat program, written and actively developed by our colleague Roberto Tejero (Universita de Valencia), is routinely used in the Montelione lab for the analysis, conversion, and manipulation of coordinate and constraint files for protein structure determination. The program is also an integral component of the Protein Structure Validation Software package [1] used across the NESG consortium. <br />
<br />
The current version of the software is PdbStat 5.1 (July, 2008). There is no separate publication for PdbStat; we cite this program using Bhattacharya et al, 2007 [1]. Also, there is no official manual for the program. Below are a number of basic stand-alone PdbStat commands and common uses. Going forward, we can continue to add to this document as more applications are developed. <br />
<br />
Anyone interested in obtaining the latest version of PdbStat can contact Roberto at: [mailto:roberto.tejero@uv.es roberto.tejero@uv.es] <br> <br />
<br />
== '''Commands and Applications''' ==<br />
<br />
==== Starting the program ====<br />
<br />
<tt>pdbstat</tt><br />
<br />
==== Menu of Commands ====<br />
<br />
<tt>menu</tt> gives a list of commands and keywords recognized by PdbStat (also get this using the command: <tt>help command</tt>) <br />
<br />
Here are the recognized PdbStat commands for version 5.1: <br />
<pre>PdbStat&gt; menu<br />
<br />
Commands/Keywords currently recognized by PDBSTAT<br />
<br />
The commands/keywords currently recognized by PDBSTAT are given below. <br />
Type "help &lt;command&gt;" for more information on each PDBSTAT function.<br />
<br />
align analyze author bye class<br />
clear close chain check chiral<br />
delete debug dump energy evaluate <br />
find fit fix get help<br />
history hydrogen hyper kabsch ident/chain<br />
info initialize log/change load menu<br />
missing order phi quit rama<br />
read relax reset restore rotate<br />
save see set show rmsd<br />
to trans version what write<br />
<br />
homology commands: <br />
get change homology homa<br />
<br />
Other kind/commands/keywords of help<br />
codes(amino codes) amino(amino geometry)&lt;/onlyinclude&gt;</pre> <br />
==== Help documents for specific commands ====<br />
<br />
Each command has a short help document describing its usage and options. <br> Syntax:<br> help {topic} {subtopic} <br />
<br />
Example: <onlyinclude>PdbStat> help read<br />
<br />
Read<br />
Syntax: rea[d] {<arg1>} {<arg2>} {<arg3>}<br />
<br />
arg1 can be: coo[rdinates] or con[straints] or seq[uence]<br />
arg2 can be: pdb or con[gen] or cha[rmm] or dis[man]<br />
arg3 can be: the name of the file.<br />
<br />
Examples: read coord pdb filename.pdb<br />
read seq filename<br />
read cons congen filename<br />
read cons diana filename<br />
<br />
Read coordinates/constraints/sequence in specified format from file. This <br />
command is able to open the file read it locating the number of structures <br />
stored and then read them.<br />
<br />
The user needs to type only <read> then a interactive cycle with questions<br />
begins. The program offers the different formats available, so after read<br />
is typed the program offers <coords/cons/seq> and then after that the<br />
program offers the different formats <pdb,disman,....></onlyinclude> <br />
<br />
<br> <br />
<br />
==== Preparation of final CNS coordinates for PDB deposition ====<br />
<br />
Before we deposit our final coordinates we would like to order our models in order of lowest conformation energy. Also, we want to perform backbone superposition of the coordinates using ordered [S(phi) + S(psi) &gt; 1.8] residues only. Finally, we want to rotate the ensemble to a desired orientation, which will be the orientation that appears when a user downloads our coordinates from the Protein Data Bank (www.rcsb.org). The final coordinates are saved in the original (CNS) format and IUPAC format for RPF analysis [2]. <br />
<br />
To prepare the final coordinates file for PDB deposition use the following protocol: <br />
<br />
PdbStat: <br> <onlyinclude><br />
rea coo pdb [filename] #read file with concatenated CNS pdb files<br />
all #select all the models<br />
class #classify the models by energy<br />
order 0.9 #determine ordered residues; phi/psi cut-off 0.9<br />
rmsd best backbone #backbone rmsd<br />
[return] #creates an rmsd output file<br />
write coo pdb [overlayed file] #write overlayed coordinates</onlyinclude> <br />
<br />
Next, open the overlayed coordinates in Molmol and get the desired rotation. Use <tt>writetransform</tt> command to write the rotation matrix to a file. <br> Back in PdbStat: <br> <onlyinclude><br />
rea coo pdb [overlayed.pdb] #read the overlayed coordinates (all models)<br />
rotate file [filename] #apply rotation matrix<br />
write coo pdb [final.pdb] #write new coordinates<br />
to iupac #converts atom nomenclature to IUPAC<br />
write coo pdb [final_iupac.pdb] #write IUPAC coordinates for RPF analysis</onlyinclude> <br />
<br />
==== Selecting specific models / residues / atoms. ====<br />
<br />
A powerful option in PdbStat is the ability to select specific residues and/or atoms for further analysis. This is extremely useful for superposition and RMSD evaluation of selected residues/atoms. <br> Syntax and Examples: <br> <br />
<pre>#syntax<br />
sel[ect] {model(s)} {residue(s)} {atom(s)} <br />
#select backbone atoms of residues 5-25,30-50 in models 1,3-5,7-10<br />
sele 1,3-5,7-10 5-25,30-50 backbone <br />
#select N,C,CA,O atoms of residues 5-50 in all models <br />
sele * 5-50 n,c,ca,o <br />
#combine selection with superposition <br />
sele * 5-50,60-85 *<br />
rmsd sele backbone<br />
</pre> <br />
==== Conversion of coordinate and constraint formats ====<br />
<br />
In the course of a structure refinement we regularly have to convert between different coordinate and constraint formats for different structure programs (i.e., CYANA, XPLOR/CNS, ECEPP). We routinely use PdbStat for this. <br> Examples:<br> <br />
<pre># converting CYANA to XPLOR/CNS coordinates:<br />
rea coor pdb [CYANA.pdb] #read CYANA models (all)<br />
to xplor #converts to xplor; fixes stereospecfic labels <br />
write #answer questions<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns.pdb<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ coor<br />
&gt; WRITER: ... backbone, heavy, full set? (back/heavy/all/select): all<br />
&gt; WRITER: What model&nbsp;?_&nbsp;: all<br />
&gt; WRITER: -- All models to be written <br />
&gt; COORD_writer: Format (pdb/congen/RasMol)&nbsp;?&nbsp;: pdb <br />
# converting CYANA to XPLOR/CNS distance constraints:<br />
rea coor pdb [CYANA.pdb] #read CYANA models; you have to read in a pdb or sequence file before the constraints<br />
rea cons cyana [CYANA.upl] #read CYANA upls file<br />
write #write to CNS format; add 10% to upper bound; make lower bound van der Waals (1.8 A)<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_noe.tbl <br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ cons<br />
&gt; WRITER_constr: Output format <br />
[congen|discover|ecepp|disman|dyana|cyana|diana|xplor|cns]?&nbsp;: cns<br />
&gt; XPLOR_writer: percentage range for upper bound (upp+range)?: 10<br />
&gt; XPLOR_writer: range for lower bound (low-range)?: vdw<br />
&gt; XPLOR_writer: ... writing ... wait <br />
# converting CYANA to XPLOR/CNS dihedral constraints:<br />
rea aco cyana [CYANA.aco]<br />
write<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_dihe.tbl<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ aco <br />
&gt; WRITER_constr: Output format (congen | discover | impact | disman | diana | xplor | cns)?&nbsp;: cns</pre> <br />
==== Constraint Violations ====<br />
<br />
One can analyze constraint violations using PdbStat. It is best to convert the coordinates and constraints to IUPAC format; this is the approach used internally within PSVS. <br> Commands: <onlyinclude><br />
rea coo pdb [filename]<br />
to iupac #convert coordinates to IUPAC format<br />
rea cons [format] [filename] #read distance constraints; specify format<br />
noe to iupac #convert distance constraints to IUPAC<br />
rea aco [format] [filename] #read dihedral constraints; specify format<br />
set cutu 0.2 #set cut-off for distance violations to 0.2 A<br />
see viol noe [sum/ave/center] #see noe violations using sum/average/center averaging<br />
see cutaco 1 #set cut-off for dihedral violations to 1 deg.<br />
see viol aco #see dihedral violations above threshold</onlyinclude> <br />
<br />
==== Sorting of Distance Constraints ====<br />
<br />
There are a number of options in PdbStat for sorting or culling distance constraints. <br> <onlyinclude><br />
cons clean #keep conformationally-restricting constraints; also removes duplicates<br />
noe analysis #NOE statistics (as in PSVS)<br />
noe delete intra #delete all intra NOE constraints<br />
noe keep long #keep only long range (||i-j|| >/= 5) NOE constraints<br />
noe keep ilv #keep NOE constraints consistent with ILV labeling</onlyinclude> <br />
<br />
==== Commands for Obtaining Various Metrics ====<br />
<br />
The “eval”, “show”, and “see” commands allow one to evaluate several types of metrics in a structure or ensemble. <br> <onlyinclude><br />
eval [procheck/rama] #get Procheck/Ramachandran statistics for model(s)<br />
eval dist * 68 sg 119 nd1 #get 68-SG to 119-ND1 distance across all models<br />
eval [phi || psi || omega] 1 #get phi/psi/omega torsion angles in model 1<br />
see lib [residue] #see library definitions for residue type<br />
select 1 119 * #use select and see in tandem<br />
see coo #coordinates for residue 119 in model 1</onlyinclude> <br />
<br />
==== Other Functions ====<br />
<br />
<onlyinclude><br />
# renumbering / resetting residue numbering<br />
reset * 10 #sets first residue in file to 10<br />
reset * #resets coordinates to original<br />
# ordering ensemble using FindCore algorithm [3]:<br />
find [ -bb || -heavy || -all || -noe ]<br />
# contact map generation based on coordinates or constraints<br />
contact<br />
> DRAW_cntct: from coordinates or constraints (coor/cons) ?:_ coor<br />
> MAIN_cntct: What model do you want (1-20) or average ?_ :ave<br />
> do_average_coords(): Making average for backbone atoms <br />
> do_average_coords(): Calculating center of masses <br />
> do_average_coords(): Calling optimal rotation for backbone <br />
> do_average_coords(): Calc. average coordinates backbone <br />
> MAIN_cntct: distance cutoff ?:_ 4.5<br />
> MAIN_cntct: atom type (hydr, heavy, all) ?:_ hydr #writes a postscript file</onlyinclude> <br />
<br />
<br> <br />
<br />
== <span style="font-weight: bold;">Updates</span> ==<br />
<br />
==== Version 5.4<br> ====<br />
<br />
Several new functions have been added to PdbStat and a new version of the program (PdbStat 5.4) was released in April 2011.&nbsp; <br> <br />
<br />
== '''References''' ==<br />
<br />
[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795. ]<br>[http://www.ncbi.nlm.nih.gov/pubmed/15701001?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=4 2. Huang, Y. J., Powers, R., and Montelione, G.T. (2005) Protein NMR Recall, Precision, and F-measure scores (RPF scores): structure quality assessment measures based on information retrieval statistics. ''J. Am. Chem. Soc. 127'', 1665-1674. ]<br>[http://www3.interscience.wiley.com/journal/110445756/abstract 3. Snyder, D. A. and Montelione, G. T. (2005) Clustering algorithms for identifying core atom sets and for assessing the precision of protein structure ensembles. ''Proteins 59'', 673-686. ]<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=PdbStat&diff=3836
PdbStat
2011-10-05T13:54:44Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
The PdbStat program, written and actively developed by our colleague Roberto Tejero (Universita de Valencia), is routinely used in the Montelione lab for the analysis, conversion, and manipulation of coordinate and constraint files for protein structure determination. The program is also an integral component of the Protein Structure Validation Software package [1] used across the NESG consortium. <br />
<br />
The current version of the software is PdbStat 5.1 (July, 2008). There is no separate publication for PdbStat; we cite this program using Bhattacharya et al, 2007 [1]. Also, there is no official manual for the program. Below are a number of basic stand-alone PdbStat commands and common uses. Going forward, we can continue to add to this document as more applications are developed. <br />
<br />
Anyone interested in obtaining the latest version of PdbStat can contact Roberto at: [mailto:roberto.tejero@uv.es roberto.tejero@uv.es] <br> <br />
<br />
== '''Commands and Applications''' ==<br />
<br />
==== Starting the program ====<br />
<br />
<tt>pdbstat</tt><br />
<br />
==== Menu of Commands ====<br />
<br />
<tt>menu</tt> gives a list of commands and keywords recognized by PdbStat (also get this using the command: <tt>help command</tt>) <br />
<br />
Here are the recognized PdbStat commands for version 5.1: <br />
<pre>PdbStat&gt; menu<br />
<br />
Commands/Keywords currently recognized by PDBSTAT<br />
<br />
The commands/keywords currently recognized by PDBSTAT are given below. <br />
Type "help &lt;command&gt;" for more information on each PDBSTAT function.<br />
<br />
align analyze author bye class<br />
clear close chain check chiral<br />
delete debug dump energy evaluate <br />
find fit fix get help<br />
history hydrogen hyper kabsch ident/chain<br />
info initialize log/change load menu<br />
missing order phi quit rama<br />
read relax reset restore rotate<br />
save see set show rmsd<br />
to trans version what write<br />
<br />
homology commands: <br />
get change homology homa<br />
<br />
Other kind/commands/keywords of help<br />
codes(amino codes) amino(amino geometry)&lt;/onlyinclude&gt;</pre> <br />
==== Help documents for specific commands ====<br />
<br />
Each command has a short help document describing its usage and options. <br> Syntax:<br> help {topic} {subtopic} <br />
<br />
Example: <onlyinclude>PdbStat> help read<br />
<br />
Read<br />
Syntax: rea[d] {<arg1>} {<arg2>} {<arg3>}<br />
<br />
arg1 can be: coo[rdinates] or con[straints] or seq[uence]<br />
arg2 can be: pdb or con[gen] or cha[rmm] or dis[man]<br />
arg3 can be: the name of the file.<br />
<br />
Examples: read coord pdb filename.pdb<br />
read seq filename<br />
read cons congen filename<br />
read cons diana filename<br />
<br />
Read coordinates/constraints/sequence in specified format from file. This <br />
command is able to open the file read it locating the number of structures <br />
stored and then read them.<br />
<br />
The user needs to type only <read> then a interactive cycle with questions<br />
begins. The program offers the different formats available, so after read<br />
is typed the program offers <coords/cons/seq> and then after that the<br />
program offers the different formats <pdb,disman,....></onlyinclude> <br />
<br />
<br> <br />
<br />
==== Preparation of final CNS coordinates for PDB deposition ====<br />
<br />
Before we deposit our final coordinates we would like to order our models in order of lowest conformation energy. Also, we want to perform backbone superposition of the coordinates using ordered [S(phi) + S(psi) &gt; 1.8] residues only. Finally, we want to rotate the ensemble to a desired orientation, which will be the orientation that appears when a user downloads our coordinates from the Protein Data Bank (www.rcsb.org). The final coordinates are saved in the original (CNS) format and IUPAC format for RPF analysis [2]. <br />
<br />
To prepare the final coordinates file for PDB deposition use the following protocol: <br />
<br />
PdbStat: <br> <onlyinclude><br />
rea coo pdb [filename] #read file with concatenated CNS pdb files<br />
all #select all the models<br />
class #classify the models by energy<br />
order 0.9 #determine ordered residues; phi/psi cut-off 0.9<br />
rmsd best backbone #backbone rmsd<br />
[return] #creates an rmsd output file<br />
write coo pdb [overlayed file] #write overlayed coordinates</onlyinclude> <br />
<br />
Next, open the overlayed coordinates in Molmol and get the desired rotation. Use <tt>writetransform</tt> command to write the rotation matrix to a file. <br> Back in PdbStat: <br> <onlyinclude><br />
rea coo pdb [overlayed.pdb] #read the overlayed coordinates (all models)<br />
rotate file [filename] #apply rotation matrix<br />
write coo pdb [final.pdb] #write new coordinates<br />
to iupac #converts atom nomenclature to IUPAC<br />
write coo pdb [final_iupac.pdb] #write IUPAC coordinates for RPF analysis</onlyinclude> <br />
<br />
==== Selecting specific models / residues / atoms. ====<br />
<br />
A powerful option in PdbStat is the ability to select specific residues and/or atoms for further analysis. This is extremely useful for superposition and RMSD evaluation of selected residues/atoms. <br> Syntax and Examples: <br> <br />
<pre>#syntax<br />
sel[ect] {model(s)} {residue(s)} {atom(s)} <br />
#select backbone atoms of residues 5-25,30-50 in models 1,3-5,7-10<br />
sele 1,3-5,7-10 5-25,30-50 backbone <br />
#select N,C,CA,O atoms of residues 5-50 in all models <br />
sele * 5-50 n,c,ca,o <br />
#combine selection with superposition <br />
sele * 5-50,60-85 *<br />
rmsd sele backbone<br />
</pre> <br />
==== Conversion of coordinate and constraint formats ====<br />
<br />
In the course of a structure refinement we regularly have to convert between different coordinate and constraint formats for different structure programs (i.e., CYANA, XPLOR/CNS, ECEPP). We routinely use PdbStat for this. <br> Examples:<br> <br />
<pre># converting CYANA to XPLOR/CNS coordinates:<br />
rea coor pdb [CYANA.pdb] #read CYANA models (all)<br />
to xplor #converts to xplor; fixes stereospecfic labels <br />
write #answer questions<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns.pdb<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ coor<br />
&gt; WRITER: ... backbone, heavy, full set? (back/heavy/all/select): all<br />
&gt; WRITER: What model&nbsp;?_&nbsp;: all<br />
&gt; WRITER: -- All models to be written <br />
&gt; COORD_writer: Format (pdb/congen/RasMol)&nbsp;?&nbsp;: pdb <br />
# converting CYANA to XPLOR/CNS distance constraints:<br />
rea coor pdb [CYANA.pdb] #read CYANA models; you have to read in a pdb or sequence file before the constraints<br />
rea cons cyana [CYANA.upl] #read CYANA upls file<br />
write #write to CNS format; add 10% to upper bound; make lower bound van der Waals (1.8 A)<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_noe.tbl <br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ cons<br />
&gt; WRITER_constr: Output format <br />
[congen|discover|ecepp|disman|dyana|cyana|diana|xplor|cns]?&nbsp;: cns<br />
&gt; XPLOR_writer: percentage range for upper bound (upp+range)?: 10<br />
&gt; XPLOR_writer: range for lower bound (low-range)?: vdw<br />
&gt; XPLOR_writer: ... writing ... wait <br />
# converting CYANA to XPLOR/CNS dihedral constraints:<br />
rea aco cyana [CYANA.aco]<br />
write<br />
&gt; WRITER: Output file name&nbsp;?:_ 4cns_dihe.tbl<br />
&gt; WRITER: Coords, constraints, aco or sequence file?&nbsp;:_ aco <br />
&gt; WRITER_constr: Output format (congen | discover | impact | disman | diana | xplor | cns)?&nbsp;: cns</pre> <br />
==== Constraint Violations ====<br />
<br />
One can analyze constraint violations using PdbStat. It is best to convert the coordinates and constraints to IUPAC format; this is the approach used internally within PSVS. <br> Commands: <onlyinclude><br />
rea coo pdb [filename]<br />
to iupac #convert coordinates to IUPAC format<br />
rea cons [format] [filename] #read distance constraints; specify format<br />
noe to iupac #convert distance constraints to IUPAC<br />
rea aco [format] [filename] #read dihedral constraints; specify format<br />
set cutu 0.2 #set cut-off for distance violations to 0.2 A<br />
see viol noe [sum/ave/center] #see noe violations using sum/average/center averaging<br />
see cutaco 1 #set cut-off for dihedral violations to 1 deg.<br />
see viol aco #see dihedral violations above threshold</onlyinclude> <br />
<br />
==== Sorting of Distance Constraints ====<br />
<br />
There are a number of options in PdbStat for sorting or culling distance constraints. <br> <onlyinclude><br />
cons clean #keep conformationally-restricting constraints; also removes duplicates<br />
noe analysis #NOE statistics (as in PSVS)<br />
noe delete intra #delete all intra NOE constraints<br />
noe keep long #keep only long range (||i-j|| >/= 5) NOE constraints<br />
noe keep ilv #keep NOE constraints consistent with ILV labeling</onlyinclude> <br />
<br />
==== Commands for Obtaining Various Metrics ====<br />
<br />
The “eval”, “show”, and “see” commands allow one to evaluate several types of metrics in a structure or ensemble. <br> <onlyinclude><br />
eval [procheck/rama] #get Procheck/Ramachandran statistics for model(s)<br />
eval dist * 68 sg 119 nd1 #get 68-SG to 119-ND1 distance across all models<br />
eval [phi || psi || omega] 1 #get phi/psi/omega torsion angles in model 1<br />
see lib [residue] #see library definitions for residue type<br />
select 1 119 * #use select and see in tandem<br />
see coo #coordinates for residue 119 in model 1</onlyinclude> <br />
<br />
==== Other Functions ====<br />
<br />
<onlyinclude><br />
# renumbering / resetting residue numbering<br />
reset * 10 #sets first residue in file to 10<br />
reset * #resets coordinates to original<br />
# ordering ensemble using FindCore algorithm [3]:<br />
find [ -bb || -heavy || -all || -noe ]<br />
# contact map generation based on coordinates or constraints<br />
contact<br />
> DRAW_cntct: from coordinates or constraints (coor/cons) ?:_ coor<br />
> MAIN_cntct: What model do you want (1-20) or average ?_ :ave<br />
> do_average_coords(): Making average for backbone atoms <br />
> do_average_coords(): Calculating center of masses <br />
> do_average_coords(): Calling optimal rotation for backbone <br />
> do_average_coords(): Calc. average coordinates backbone <br />
> MAIN_cntct: distance cutoff ?:_ 4.5<br />
> MAIN_cntct: atom type (hydr, heavy, all) ?:_ hydr #writes a postscript file</onlyinclude> <br />
<br />
<br> <br />
<span style="font-weight: bold;">Updates</span><br> ==<br />
<br />
==== Version 5.4<br> ====<br />
<br />
Several new functions have been added to PdbStat and a new version of the program (PdbStat 5.4) was released in April 2011.&nbsp; <br><br />
<br />
== '''References''' ==<br />
<br />
[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795. ]<br>[http://www.ncbi.nlm.nih.gov/pubmed/15701001?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=4 2. Huang, Y. J., Powers, R., and Montelione, G.T. (2005) Protein NMR Recall, Precision, and F-measure scores (RPF scores): structure quality assessment measures based on information retrieval statistics. ''J. Am. Chem. Soc. 127'', 1665-1674. ]<br>[http://www3.interscience.wiley.com/journal/110445756/abstract 3. Snyder, D. A. and Montelione, G. T. (2005) Clustering algorithms for identifying core atom sets and for assessing the precision of protein structure ensembles. ''Proteins 59'', 673-686. ]<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=TALOS&diff=3605
TALOS
2010-01-07T17:03:00Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
TALOS (Torsion Angle Likelihood Obtained from Shift and sequence similarity) is a database system for empirical prediction of <tt>phi</tt> and <tt>psi</tt> backbone torsion angles from five kinds (HA, CA, CB, CO, N) of chemical shifts for a given protein sequence [1].&nbsp; In 2009, the Bax laboratory released a new and improved version of the program called TALOS+ [2].<br> <br />
<br />
For detailed information please check the [http://spin.niddk.nih.gov/NMRPipe/talos/ TALOS] and [http://spin.niddk.nih.gov/bax/software/TALOS+/index.html TALOS+] web sites.&nbsp; For installation questions and other support, you can also e-mail [mailto:shenyang@niddk.nih.gov Yang Shen]. <br />
<br />
== '''Using TALOS with CYANA''' ==<br />
<br />
=== '''Protocol at UB''' ===<br />
<br />
#Create a subdirectory (for example, <tt>structure/cyana21/talos</tt>) and copy the latest sequence and atom list files there. It is convenient to have them named <tt>XXXX.seq</tt> and <tt>XXXX.prot</tt>, where <tt>XXXX</tt> is an NESG target ID or other protein name. When using CARA, export the chemical shifts as an atom list file in this directory. <br />
#Create and init.cya in this directory as described in "[[NESG:CYANAInitFile|Creating an init.cya file for CYANA 2.1]]" or copy a previously used file. <br />
#Start CYANA and type:<br />
<br />
read prot XXXX.prot<br />
taloslist XXXX<br />
<br />
#This will create the TALOS input file <tt>XXXX.tab</tt>. In a UNIX shell run <tt>talos.tcl -in XXXX.tab</tt>. During the database search, a series of files <tt>pred/res*.tab</tt> will be created. Each one of these files tallies the 10 best database matches for a given residue in the target protein. Before exiting, <tt>talos.tcl</tt> calls <tt>vina.tcl</tt> to create a file <tt>pred.tab</tt>, which includes an initial summary of the prediction results. The database search will typically take about 10-60 sec per residue in the target. <br />
#In a UNIX shell run <tt>rama.tcl -in XXXX.tab</tt>. Here you can examine <tt>phi</tt> and <tt>psi</tt> distributions, choose database matches to be used in calculating predictions, and classify prediction results as <tt>Good</tt>, <tt>Ambiguous</tt> or <tt>Unclassified</tt> / <tt>New</tt>. See below for the guidelines for classifying prediction. Save your modifications in a new file, for example, <tt>talos.tab</tt>. <br />
#Start CYANA and type:<br />
<br />
talosaco pred #or "talos.tab" -- use the appropriate filename<br />
write aco talos.aco<br />
<br />
See also the <tt>~/demo/details/TalosAngleRestraints.cya</tt> example script in your local CYANA 2.1 installation. <br />
<br />
<br> <br />
<br />
=== '''Manual Refinement of TALOS Predictions''' ===<br />
<br />
Guidelines for refining the TALOS output: <br />
<br />
*Classify prediction as <tt>Good</tt> only if <br />
**All 10 best database matches fall in a "consistent" region of the Ramachandran map <br />
**Or 9 out of 10 best database matches fall in a consistent region with <tt>phi &lt; 0</tt>, and the one outlier also lies in <tt>phi &lt; 0</tt> half of the map <br />
**Or 9 out of 10 of the best database matches fall in a consistent region with <tt>phi &gt; 0</tt> <br />
*Accept predictions which are classified as <tt>Good</tt>, whose residues are in beta-sheets or helices according to CSI (excluding the first and the last residue of a secondary structure element).<br />
<br />
For ''de novo'' structure determination it is recommended to take the automatically generated TALOS constraints. Angular constraints outside of secondary structure elements (as determined by CSI) can be commented out in the <tt>talos.aco</tt> file. <br />
<br />
During structure refinement you can refine TALOS predictions against a preliminary structure. <br />
<pre>vina.tcl -in XXXX.tab -ref XXXX.pdb -auto</pre> <br />
and <br />
<pre>rama.tcl -in XXXX.tab -ref XXXX.pdb</pre> <br />
<br> The <tt>XXXX.pdb</tt> file '''must''' have only one conformer. Thus, you may need to analyze the angle distributions in a molecular graphics package (e.g. MOLMOL). <br />
<br />
<br> <br />
<br />
{| border="1"<br />
|-<br />
| Element <br />
| PHI <br />
| PSI<br />
|-<br />
| α-helix <br />
| -60 <br />
| -45<br />
|-<br />
| β-sheet <br />
| -140 <br />
| 135<br />
|}<br />
<br />
<br> <br />
<br />
=== '''Description of talosaco.cya Macro''' ===<br />
<br />
Invoked as: <br />
<pre>talosaco file [factor [width]]</pre> <br />
Here <tt>file</tt> is the TALOS prediction output, <tt>width</tt> is the threshold minimum width for <tt>PHI/PSI</tt> angle distributions, and <tt>factor</tt> is used to scale the width of a distribution when creating an angle constraint. Both <tt>width</tt> and <tt>factor</tt> arguments are optional. By default, <tt>width=20.0</tt> and <tt>factor=2.0</tt>. <br />
<br />
This macro will create angle constraints for a given residue only if the prediction is classified as "Good" and the residue is not a proline. <br />
<br />
<br> <br />
<br />
== '''Using TALOS and TALOS+ at CABM''' ==<br />
<br />
=== Preparing for a TALOS+ run ===<br />
<br />
*Make a sub-directory in your project for TALOS. <br />
*you will need the following files in your directory: <br />
*a bmrb file in 2.1 format. &nbsp;Here is an [[Media:PfR193A_062509_2.1f_4CYANA.bmrb|example]]. <br />
*[[Media:BMRBParsing.pm|BMRBParsing.pm]]:&nbsp; BMRB&nbsp;parser <br />
*[[Media:Tab4Talos.txt|Tab4Talos.pl]]:&nbsp; perl script to prepare input file for TALOS <br />
*[[Media:Talos2dyana_taloserrors.txt|talos2dyana_taloserrors.pl]]:&nbsp; perl script to prepare a CYANA .aco file <br />
*Run the following command:<br />
<pre> Tab4Talos.pl [.bmrbf] [input4Talos]<br />
<br />
</pre> <br />
This make an input chemical shift list for TALOS.&nbsp; Here is an [[Media:PfR193A_4Talos.input|example]].<br> <br />
<br />
=== Running TALOS+ and making a dihedral angle constraint file<br> ===<br />
<br />
*Next run talos+:<br />
<pre> talos+ -in [input4Talos</pre> <br />
This makes a number of output files including the pred.tab.&nbsp; <br> <br />
<br />
*Next, edit the pred.tab and comment out (#) any lines that do not have the "10 Good" comment.&nbsp; <br />
*Finally, run the talos2cyana perl script to make a CYANA .aco file with only the results classified as "10 Good", and using the phi and psi errors given by TALOS.&nbsp; They user can modify this script to make his/her own error limits (i.e., +/- 20 or 30).<br />
<pre> perl talos2dyana_taloserrors.pl pred.tab [output.aco]<br />
</pre> <br />
<br> <br />
<br />
== '''References''' ==<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10212987?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=3 <font face="Arial,Helvetica">1.&nbsp;&nbsp;&nbsp; Cornilescu, G., Delaglio, F. and Bax, A. (1999) </font><font face="Arial,Helvetica">Protein backbone angle restraints from searching a database for chemical shift and sequence homology. </font><font face="Arial,Helvetica">''J. Biomol. NMR 13'', 289-302.</font>] <br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19548092?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1 <font face="Arial,Helvetica">2.&nbsp;&nbsp;&nbsp; Shen, Y., </font><font face="Arial,Helvetica">Delaglio, F., Cornilescu, G. and Bax, A. (2009) </font><font face="Arial,Helvetica">TALOS+: A hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. </font><font face="Arial,Helvetica">''J. Biomol. NMR&nbsp;44'', 213-223</font>] <br />
<br />
<br> <br />
<br />
<br><br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=File:Talos2dyana_taloserrors.txt&diff=3604
File:Talos2dyana taloserrors.txt
2010-01-07T17:01:44Z
<p>Jma: </p>
<hr />
<div></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=File:Tab4Talos.txt&diff=3603
File:Tab4Talos.txt
2010-01-07T17:01:32Z
<p>Jma: </p>
<hr />
<div></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=PSVS&diff=3602
PSVS
2010-01-07T16:57:08Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
[http://www-nmr.cabm.rutgers.edu/PSVS/ PSVS] (Protein Structure Validation Server) systematically evaluates the quality of protein structures, and reports a large set of quality scores and constraint analyses [1]. PSVS defines a set of standard quality scores and constraint analyses that are reported for each structure. In addition to experimental constraints, this set encompasses a number of parameters evaluating different aspects of structure quality, including fold and packing, local residue separation, deviations of bond length and bond angles, backbone and side-chain torsion angle conformation, and atomic overlaps. Quality scores are reported based on calibration with a set of high-resolution X-ray crystal structures. A Z-score for the query structure, calculated for the score from each tool, provides an evaluation of the quality of the structure compared to this set of X-ray crystal structures. This server returns a concise validation report that includes a standard set of graphs and tables.&nbsp; <br> <br />
<br />
<br> <br />
<br />
== '''Running the PSVS server''' ==<br />
<br />
Once the structure is available, go to the [http://www-nmr.cabm.rutgers.edu/PSVS/ PSVS server] and upload the input files as described in that page (Figure 1).<br> <br />
<br />
*PDB coordinates: atom names can be in PDB format, DYANA/CYANA or CNS/XPLOR format <br />
*Peak lists (optional, for RPF scores check): separate peak lists from <sup>15</sup>N-resolved NOESY and <sup>13</sup>C-resolved NOESY. <br />
*Chemical shift (optional, for constraints violation and RPF scores check): BMRB 2.1 or 3.1 format <br />
*Constraints (optional, for constraints violation check): can be in CYANA or XPLOR/CNS format.<br />
<br />
<br> <br />
<br />
===== Figure 1:&nbsp; PSVS 1.4 input page =====<br />
<br />
[[Image:PSVS1.4 titlepage.png|800px]] <br />
<br />
<br> <br />
<br />
== '''PSVS Output''' ==<br />
<br />
One will get an e-mail notice a few minutes after PSVS submisson, and following the link in the e-mail will lead to the PSVS results (Figure 2).&nbsp; Various outputs can be accessed from tabs in the left margin, and a zipped file of the entire analysis can be downloaded.<br> <br />
<br />
Typical PSVS&nbsp;analyses include: <br />
<br />
*Secondary Structure Elements <br />
*Ordered residues <br />
*RMSD values for all residues and ordered residues. <br />
*Ramachandran Plot Summary for ordered residues from Procheck <br />
**Global quality scores for [http://nihserver.mbi.ucla.edu/Verify_3D/ Verify3D], [http://www.came.sbg.ac.at/Services/prosa.html ProsaII], [http://www.biochem.ucl.ac.uk/~roman/procheck/procheck.html Procheck] (phi-psi), Procheck (all) and [http://molprobity.biochem.duke.edu/ MolProbity] Clashscore. It is required that one should never deposit a structure to the PDB with Z scores &lt; -5 for Procheck scores and Molprobity clashscores. General, Z szores &gt; -3 for both Procheck G factor and Molprobity Clashscore are recommended. <br />
**RPF Scores (optional). A good structure should have R &gt; 90%, P &gt; 90%, F&gt; 90% and DP &gt; 70%.<br />
<br />
<br> <br />
<br />
===== Figure 2:&nbsp; PSVS 1.4 output page =====<br />
<br />
[[Image:PSVS1.4_outputpage_new2.png|1000px]]<br> <br />
<br />
<br> <br />
<br />
== '''References''' ==<br />
<br />
[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795.]</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=File:PSVS1.4_outputpage_new2.png&diff=3601
File:PSVS1.4 outputpage new2.png
2010-01-07T16:56:08Z
<p>Jma: </p>
<hr />
<div></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=File:Example_NMR_rosetta_refinement.tar.gz&diff=3600
File:Example NMR rosetta refinement.tar.gz
2010-01-07T16:52:18Z
<p>Jma: </p>
<hr />
<div></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=PSVS&diff=3599
PSVS
2010-01-07T16:41:14Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
[http://www-nmr.cabm.rutgers.edu/PSVS/ PSVS] (Protein Structure Validation Server) systematically evaluates the quality of protein structures, and reports a large set of quality scores and constraint analyses [1]. PSVS defines a set of standard quality scores and constraint analyses that are reported for each structure. In addition to experimental constraints, this set encompasses a number of parameters evaluating different aspects of structure quality, including fold and packing, local residue separation, deviations of bond length and bond angles, backbone and side-chain torsion angle conformation, and atomic overlaps. Quality scores are reported based on calibration with a set of high-resolution X-ray crystal structures. A Z-score for the query structure, calculated for the score from each tool, provides an evaluation of the quality of the structure compared to this set of X-ray crystal structures. This server returns a concise validation report that includes a standard set of graphs and tables.&nbsp; <br> <br />
<br />
<br> <br />
<br />
== '''Running the PSVS server''' ==<br />
<br />
Once the structure is available, go to the [http://www-nmr.cabm.rutgers.edu/PSVS/ PSVS server] and upload the input files as described in that page (Figure 1).<br> <br />
<br />
*PDB coordinates: atom names can be in PDB format, DYANA/CYANA or CNS/XPLOR format <br />
*Peak lists (optional, for RPF scores check): separate peak lists from <sup>15</sup>N-resolved NOESY and <sup>13</sup>C-resolved NOESY. <br />
*Chemical shift (optional, for constraints violation and RPF scores check): BMRB 2.1 or 3.1 format <br />
*Constraints (optional, for constraints violation check): can be in CYANA or XPLOR/CNS format.<br />
<br />
<br> <br />
<br />
===== Figure 1:&nbsp; PSVS 1.4 input page =====<br />
<br />
[[Image:PSVS1.4 titlepage.png|800px]] <br />
<br />
<br> <br />
<br />
== '''PSVS Output''' ==<br />
<br />
One will get an e-mail notice a few minutes after PSVS submisson, and following the link in the e-mail will lead to the PSVS results (Figure 2).&nbsp; Various outputs can be accessed from tabs in the left margin, and a zipped file of the entire analysis can be downloaded.<br> <br />
<br />
Typical PSVS&nbsp;analyses include: <br />
<br />
*Secondary Structure Elements <br />
*Ordered residues <br />
*RMSD values for all residues and ordered residues. <br />
*Ramachandran Plot Summary for ordered residues from Procheck <br />
**Global quality scores for [http://nihserver.mbi.ucla.edu/Verify_3D/ Verify3D], [http://www.came.sbg.ac.at/Services/prosa.html ProsaII], [http://www.biochem.ucl.ac.uk/~roman/procheck/procheck.html Procheck] (phi-psi), Procheck (all) and [http://molprobity.biochem.duke.edu/ MolProbity] Clashscore. It is required that one should never deposit a structure to the PDB with Z scores &lt; -5 for Procheck scores and Molprobity clashscores. General, Z szores &gt; -3 for both Procheck G factor and Molprobity Clashscore are recommended. <br />
**RPF Scores (optional). A good structure should have R &gt; 90%, P &gt; 90%, F&gt; 90% and DP &gt; 70%.<br />
<br />
<br> <br />
<br />
===== Figure 2:&nbsp; PSVS 1.4 output page (partial) =====<br />
<br />
[[Image:PSVS1.4_outputpage_new.png|1000px]]<br> <br />
<br />
<br> <br />
<br />
== '''References''' ==<br />
<br />
[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795.]</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=File:PSVS1.4_outputpage_new.png&diff=3598
File:PSVS1.4 outputpage new.png
2010-01-07T16:38:03Z
<p>Jma: </p>
<hr />
<div></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Structure_Refinement_Using_CNS_Energy_Minimization_With_Explicit_Water&diff=3596
Structure Refinement Using CNS Energy Minimization With Explicit Water
2010-01-06T21:59:01Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
Crystallography &amp; NMR System (CNS) is a program has been designed to provide a flexible multi-level hierachical approach for the most commonly used algorithms in macromolecular structure determination [1]. Functions include heavy atom searching, experimental phasing (including MAD and MIR), density modification, crystallographic refinement with maximum likelihood targets, and NMR structure calculation using NOEs, J-coupling, chemical shift, and dipolar coupling data. <br />
<br />
The latest version is 1.21. Please check the website [http://cns-online.org/ http://cns-online.org] for additional information. <br />
<br />
== '''CNS Refinement Protocol''' ==<br />
<br />
The final structure from CYANA should be refined using CNS energy minimization with explicit water before PDB deposition. <br />
<br />
The common input files are: <br />
<br />
*PDB coordinates <br />
*NOE constraints <br />
*Dihedral angle constraints <br />
*Hydrogen bond constraints<br />
<br />
==== '''Converting input files from CYANA to XPLOR/CNS'''<br> ====<br />
<br />
If the final structure calculation was performed with CYANA 2.1, then the constraint and coordinate files must be first converted to XPLOR/CNS format. <br />
<br />
==== '''Using cyana2cns.cya script''' ====<br />
<br />
This procedure can be used to prepare input files to use with the WaterRefCNS script (see below). <br />
<br />
#Download [[Media:Cyana2cns.cya|cyana2cns.cya]] into directory with the final structure calculation. <br />
#Modify the <tt>read pdb</tt>, <tt>read upl</tt> and <tt>read aco</tt> lines if necessary. <br />
#Start CYANA 2.1 and run <tt>cyana2cns.cya</tt>.<br />
<br />
*All conformers will be stored in a single PDB file <tt>KKK.pdb</tt>. If you plan run water bath refinement manually, use the <tt>p2X</tt> program instead. <br />
*The resulting NOE constraint file <tt>KKK_noe.tbl</tt> will have all lower limits set to 0. If want them to be set according to VdW radii, use the <tt>r2X</tt> program instead. Experience shows that there is no significant effect on geometry and quality scores as reported by PSVS.<br />
<br />
==== '''Using programs p2X and r2X and d2X''' ====<br />
<br />
These programs were written by Alex Lemak at the University of Toronto. They are typically used to prepare input files for manual CNS water bath refinement. <br />
<br />
Go to <tt>~1_projects/targetID/structure/cns/</tt> directory and <tt>conversion_scripts</tt> sub directory, convert required input files to CNS format from CYANA format by using macro <tt>mkfil</tt>:&nbsp; <br />
<pre> p2X cyana2.pdb pref <br />
r2X found-c.upl noeupl.tbl <br />
r2X gr-hbonds.upl hbonds.tbl<br />
cat noeupl.tbl hbonds.tbl &gt; noe.tbl <br />
d2X found-c.aco aco.tbl <br />
</pre> <br />
*<tt>p2X splits conformers from a pdb file generated by CYANA 2.1 into individual pdb files (one file per conformer) and converts atom names to CNS format. The second argument "pref"</tt> specifies a common prefix name for the output pdb files. It should be no more than 4 characters long. <br />
*<tt>r2X</tt> converts distance constraints (noe/hbond) from CYANA 2.1 format to CNS format. The lower limits in the resulting constraint file are set according to VdW radii. <br />
**Distance constraints obtained from CYANA macro <tt>caliba</tt> or <tt>calibration</tt> can be used as input files. <br />
**Distance constraints obtained from CYANA macro <tt>peak calibrate</tt> should have the pseudo-atom correction run before used as input files, run following commands under CYANA: <br> <pre>read upl final.upl<br />
</pre><br />
<br />
distance correct write upl final_corrected.upl This, however, will not add corrections for multiplicity! <br />
<br />
*<tt>d2X</tt>, to run: <tt>d2X cyana2.aco cns.tbl</tt> This will convert angle constraints from CYANA format to CNS format. If you don't have any dihedral angle constrains, create an empty file.<br />
<br />
Both p2X and r2X require a translation table file [[Media:AtomtransC.tbl|atomtransC.tbl]]. <br />
<br />
If have used simplified pseudoatom names (H* instead of Q*) with option pseudo=2 in CYANA 2.1, you may first want to them before running p2X and r2X:<br> <br />
<pre>pseudo=0<br />
read upl noe.upl<br />
read pdb cyana2.pdb<br />
write upl out.upl<br />
write pdb out.pdb all<br />
</pre> <br />
Note that pseudo=0 should be set '''before''' loading PDB and constraint files. <br />
<br />
==== '''Conversion from DYANA''' ====<br />
<br />
Use CYANA macro <tt>MigrateFromDyanaCyana1.cya</tt> to convert of data files from standard CYANA1.x or DYANA nomenclature to the standard IUPAC nomenclature used by CYANA, then do then same thing as above. <br> <br />
<pre> translate dyana # use Cyana 1.x/Dyana nomenclature<br />
read seq demo-dyana.seq # read sequence from Cyana 1.x/Dyana<br />
read upl demo-dyana.upl unknown=warn # read upper distance limits from Cyana 1.x/Dyana<br />
read aco demo-dyana.aco unknown=warn # read angle restraints from Cyana 1.x/Dyana<br />
translate off # return to standard (IUPAC) nomenclature<br />
write demo-cyana.seq # save sequence in standard Cyana format<br />
write upl demo-cyana.upl # save upper distance limits in standard Cyana format<br />
write aco demo-cyana.aco # save angle restraints in standard Cyana format <br />
</pre> <br />
<br> <br />
<br />
=== '''Running CNS using WaterRefCNS script at CABM''' ===<br />
<br />
The WaterRefCNS script, developed at Rutgers University by Roberto Tejero, performs many of the above CNS water refinement protocols in an easy to use script with many user controled options. <br />
<br />
To see a list of options type the command: <br />
<pre> WaterRefCNS<br />
</pre> <br />
You will see a list of options and how the program is used. <br />
<pre> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
WaterRefCN -- a tool to launch structure refinement with water<br />
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br />
<br />
Usage: /farm/software/WaterRefinement_cns/WaterRefCNS -na NamProt -que [PBS|No] -pro [xpl|cns]<br />
<br />
** Full set of options [not all are needed for a perfect run, use defaults]<br />
<br />
-na NamProt Name of the protein NOTE: this is mandatory, no default value <br />
-qu [PBS|NO] Que system to use, one of PBS or NOQUE def NOQUE<br />
-pr [cns|xpl] Protocol to use, X-POR, CNS def cns <br />
-av [center|sum] average for the distance restraints (for CNS, XPLOR) def sum <br />
-ci n1,n2 CIS pep info (PRO res num) in comma separated list, i.e 56,89 <br />
-ss n1-n2,n3-n4 S-S bridge info in comma separated, dash-separated pairs, i.e. 2-24,30-40 <br />
-he Display this help message<br />
-hisd n1,n2 HISD pep info (HIS res num) in comma separated list, i.e 12,43<br />
-hise n1,n2 HISE pep info (HIS res num) in comma separated list, i.e 25,67<br />
-heat N number of cycles in heating stage, default is 200 <br />
-hot N number of cycles in HOT stage, default is 1000 <br />
-cool N number of cycles for cooling stage, default is 100 <br />
-sc N Scale all terms (noe, dihe, hbond) by xN times def 1 <br />
-par string Choice of nonbonded params, one of OPLSX|PARAM19|PARMALLH6|PROLSQ|CONTACT<br />
default is OPLSX<br />
-seed &lt;int&gt; Seed for random number generator, default 31415 <br />
<br />
Examples:<br />
/farm/software/WaterRefinement_cns/WaterRefCNS -na SR358 -que no -pr cns -ci 22 <br />
<br />
</pre> <br />
Given a protein name KKK, here are the input files required to run WaterRefCNS: <br />
<br />
*&nbsp; pdb coordinates (in a single file) prepared in Xplor format using PdbStat.&nbsp; The name of the file should be:&nbsp; <tt>KKK.pdb</tt> <br />
*&nbsp; an noe.tbl file in CNS format.&nbsp; You can convert CYANA a&nbsp;final.upl file using PdbStat.&nbsp; We typically add 10-15% to the upper limit and make the lower limit vdw.&nbsp; The name of this file should be:&nbsp; <tt>KKK_noe.tbl</tt> <br />
*&nbsp; a dihe.tbl file in CNS&nbsp;format.&nbsp; Again, you can convert a CYANA .aco file using PdbStat.&nbsp; The name of this file should be:&nbsp; <tt>KKK_dihe.tbl</tt> <br />
*&nbsp; an hbond.tbl file in CNS format (optional).&nbsp; The name of this file should be:&nbsp; <tt>KKK_bhond.tbl</tt><br />
<br />
<br> <br />
<br />
In order to run WaterRefCNS on the CABM-NMR cluster: <br />
<br />
#Log into hummer or master3 <br />
#go to the directory with the above 4 files <br />
#type:<br />
<pre> /farm/software/WaterRefinement_cns/WaterRefCNS -na KKK -que PBS</pre> <br />
and that's it.&nbsp; If you have some CIS residue you add <tt>-ci</tt> NUMRES to the options above. If you want to have better vdw violation, you can add <tt>-par PARAM19</tt> to the options above. <br />
<br />
'''Future work:''' <br />
<br />
Currently, the WaterRefCNS script does not support refinement with RDC's.&nbsp; <br />
<br />
<br> <br />
<br />
=== '''WaterRefCNS at SUNY Buffalo''' ===<br />
<br />
The CABM water refinement package was customized to run on computers at UB: <br />
<br />
*Fixed a bug in <tt>WaterRefCNS</tt> so that the assembled refined PDB structure file has a <tt>.pdb</tt> extension. <br />
*PBS queue submission code in <tt>WaterRefCNS</tt> modified for use on U2 cluster <br />
*Added <tt>-hisd</tt> and <tt>-hise</tt> options to use neutral histidine isoprotomers. This required modification of <tt>WaterRefCNS</tt>, <tt>cns_refine_h2o.inp</tt> and <tt>generate_h2o.inp</tt>. <br />
*Fixed a bug in the protein topology file <tt>topallhdg5.3.pro</tt>. The line <tt>atom CD1 type=CR1E charge=0.130 end</tt> in the HISE patch section was removed. <br />
*Fixed a bug that caused the order of conformers to be changed in the output PDB file by replacing the line <tt>$WATREFLIB/Agrupa *.pdb &gt; All_${Name}_cns.pdb</tt> with <tt>$WATREFLIB/Agrupa `ls resa_?.pdb&nbsp;; ls resa_??.pdb` &gt; All_${Name}_cns.pdb</tt> in the <tt>WaterRefCNS</tt> script.<br />
<br />
The modified package is installed in <tt>/nsm/chem/cen2/HTP2/3_src/WaterRefinement_cns</tt> on the local server and in <tt>/san/projects1/szypersk/src/WaterRefinement_cns</tt> on the U2 Linux cluster. Make sure that this directories is included in your path on <tt>spins*</tt> workstations and U2, respectively. <br />
<br />
For reference, the download location is here: [[Media:WaterRefinement_UB.tar.Z|WaterRefinement_UB.tar.Z]] <br />
<br />
The required files are: <br />
<br />
*PDB coordinates (with atom names in XPLOR/CNS format) <br />
*NOE constraints (in XPLOR/CNS format) <br />
*Dihedral angle constraints (in XPLOR/CNS format) <br />
*Hydrogen bond constraints (in XPLOR/CNS format)<br />
<br />
Assuming that your coordinate file is called <tt>KKK.pdb</tt>, the constraint files should be named <tt>KKK_noe.tbl</tt>, <tt>KKK_dihe.tbl</tt> and <tt>KKK_hbond.tbl</tt>. If you don't have dihedral angle and/or hydrogen bond constraints, then create the corresponding empty files. <br />
<br />
You may need to add a line like <tt>nassign=1000</tt> at the top of the constraint files. The value should be equal to or greater than the number of constraints in the file. <br />
<br />
WaterRefCNS assumes "sum of r<sup>-6</sup>" averaging by default. When using "center"-averaged UPLs (e.g. from <tt>caliba</tt>) add <tt>-av center</tt> when starting WaterRefCNS. For information on averaging conventions for calibration in CYANA see [[NOE Calibration Using CYANA|NOE Calibration in CYANA]] <br />
<br />
It is recommended to run it on U2 Linux cluster. Examples: <br />
<br />
#<pre>WaterRefCNS -na KKK -que PBS</pre> <br />
#<pre>WaterRefCNS -na KKK -que PBS -ci 21,49</pre>residues 21 and 49 are cis-Pro <br />
#<pre>WaterRefCNS -na KKK -que PBS -hise 40,82 -hisd 32,65</pre>residues 40 and 82 are ε-protonated neutral His <br> residues 32 and 65 are δ-protonated neutral His <br />
#<pre>WaterRefCNS -na KKK -que PBS -av center </pre>for use with center-averaged calibration<br />
<br />
To run on <tt>spins*</tt> Linux workstations without queue system use <tt>-que NO</tt> option instead of <tt>-que PBS</tt> option. The default is no queue system, so you can omit the <tt>-que</tt> option altogether. <br />
<br />
==== '''Running WaterRefCNS''' ====<br />
<br />
#Convert the coordinate and constraint files as described above. <br />
#Copy the converted files into a CNS working directory on the U2 cluster or a workstation (e.g. <tt>structure/cns/calc1</tt>). <br />
#Run the <tt>WaterRefCNS</tt> with the proper arguments. <br />
#If the calculation was successful, the refined coordinate file will be stored in the <tt>refinedPDB</tt> subdirectory. Check the <tt>BeSureToREADME</tt> for details.<br />
<br />
=== '''Running CNS "manually"''' ===<br />
<br />
Go to <tt>~1_projects/targetID/structure/cns/</tt> directory and <tt>cns_scripts</tt> sub directory, follow the procedures as describe below. <br />
<br />
'''1.&nbsp; Generating MTF topology file from a PDB file. '''<br>&nbsp;&nbsp;&nbsp; modify the header of the input file <tt>generate_mtf.inp</tt> and then run <tt>cns &lt; generate_mtf.inp &gt; mtf.log</tt>. <br />
<br />
*Modify input files path and name base on individual target as follows:<br />
<pre> ...<br />
{in} pdb_file="/san/user/gliu2/u2/cns/cns/convertion_scripts/ufc1_1.pdb";<br />
{in} param_file="/san/user/gliu2/u2/cns/cns/cns_scripts/parallhdg5.3C.pro";<br />
{in} topol_file="/san/user/gliu2/u2/cns/cns/cns_scripts/topallhdg5.3.pro";<br />
{in} plink_file="/san/user/gliu2/u2/cns/cns/cns_scripts/protein.link";<br />
{out} struct_file="ufc1_1.mtf"; <br />
...<br />
</pre> <br />
*Requires these files: <br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file <br />
*[[Media:Protein.link|protein.link]]: CNS peptide linkage definition file <br />
*For <tt>cis-Proline</tt>, define the residue Id of the residue prior to <tt>cis-Proline</tt> by additional lines in <tt>generate_mtf.inp</tt> before the WRITE command<br />
<pre> patch cisp<br />
reference=nil=( resid 90 )<br />
end <br />
</pre> <br />
*If you get an error message on the atom type, you also can try&nbsp;<br />
<pre> patch cipp<br />
reference=nil=( resid 90 )<br />
end </pre> <br />
*For neutral Nε- and Nδ-protonated histidines add these lines in <tt>generate_mtf.inp</tt> before the WRITE command<br />
<pre> patch hise<br />
reference=nil=( resid 40 )<br />
end<br />
patch hisd<br />
reference=nil=( resid 65 )<br />
end </pre> <br />
*For dimer refinement, add a line containing only <tt>TER</tt> between two monomer unit coordinates in the PDB conformer that is defined in <tt>generate_mtf_cis.inp</tt> as input file.<br />
<br />
'''<br>''' <br />
<br />
'''2.&nbsp; Rebuilding hydrogen atom positions for each structure. '''<br>&nbsp;&nbsp;&nbsp; Modify input file <tt>rebuild.inp</tt> and then run <tt>cns &lt; rebuild.inp &gt; rebuild.log</tt> <br />
<br />
*Modify input files path and name base on induvidual target as follows:&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;<br />
<pre> ...<br />
evaluate ($mtf_file="ufc1_1.mtf")<br />
evaluate ($pdbname_in="../convertion_scripts/ufc1")<br />
evaluate ($pdbname_out="ufc1_rb")<br />
evaluate ($number_of_struct= 20 )<br />
...<br />
evaluate ($topol_p_file="/san/user/gliu2/u2/cns/cns/cns_scripts/topallhdg5.3.pro")<br />
evaluate ($param_p_file="/san/user/gliu2/u2/cns/cns/cns_scripts/parallhdg5.3C.pro")<br />
...<br />
</pre> <br />
*Requires these files: <br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file<br />
<br />
'''3. Refining structures with explicit water.''' <br />
<br />
*Running <tt>CNS</tt> on workstation with single processor, modify input file <tt>re_h2oc.inp</tt> and then run <tt>cns &lt; re_h2oc.inp &gt; h2o.log</tt> <br />
*Modify input files path and name base on induvidual target as follows:<br />
<pre> ...<br />
evaluate ($mtf_file="ufc1_1.mtf")<br />
evaluate ($noe_file="../noe.tbl")<br />
evaluate ($dihe_file="../aco.tbl")<br />
evaluate ($hb_file="../hbond.tbl")<br />
evaluate ($pdbname_in="ufc1_rb")<br />
evaluate ($pdbname_out="ufc1_ref")<br />
evaluate ($number_of_struct= 20 )<br />
...</pre> <br />
*Modify the weight of energy contribution part if necessary, e.g. use 2x to 10x of the following scales:<br />
<pre> scale ambi 50<br />
scale dist 50<br />
scale hbond 50<br />
end<br />
restraints dihedral<br />
scale=200<br />
end</pre> <br />
*Requires <br />
**[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file <br />
**[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file <br />
**[[Media:Topallhdg5.3.sol|topallhdg5.3.sol]]: CNS solvent topology file <br />
**[[Media:Parallhdg5.3.sol|parallhdg5.3.sol]]: CNS solvent parameter file <br />
**[[Media:Boxtyp20.pdb|boxtyp20.pdb]]: Water box <br />
**[[Media:Generate_water.cns|generate_water.cns]]: CNS protein solvation script <br />
**[[Media:Print_coorheader.cns|print_coorheader.cns]]: CNS script <br />
*Run CNS on workstation with multiple processors. <br> CNS will refine each conformer independently and in parallel, it will be much faster if 20 conformer were refined in parallal at u2.ccr.buffalo.edu.<br />
<br />
&nbsp;&nbsp;&nbsp; 1.&nbsp; Run cns_convertion as descibed above &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp; 2.&nbsp; Go to cns_scripts directory, modify input file <tt>generate_mtf_cis.inp</tt> and then run =cns &lt; generate_mtf_cis.inp = as described above &nbsp;&nbsp;&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp; 3.&nbsp; Run macro <tt>getfil</tt> <br />
<pre> cp ../convertion_scripts/noe.tbl .<br />
cp ../convertion_scripts/hbonds.tbl .<br />
cp ../convertion_scripts/aco.tbl .<br />
cp ufc1_1.mtf com/.</pre> <br />
&nbsp;&nbsp;&nbsp; 4.&nbsp; Modify input file <tt>rebuild.inp</tt> and then run <tt>cns &lt; rebuild.inp</tt> as described above. <br />
<br />
&nbsp;&nbsp;&nbsp; 5.&nbsp; Modify input file <tt>re_h2oc.inp</tt> as described above, remember to set <tt>$number_of_struct = 20</tt>. <br />
<br />
&nbsp;&nbsp;&nbsp; 6.&nbsp; Run macro <tt>subaba</tt> <br />
<br />
<code></code> <br />
<pre> #bash<br />
for i in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20<br />
do<br />
mkdir ref$i<br />
cd ref$i<br />
cp ../com/* .<br />
cp ../jan24/ufc1_rb_$i.pdb ufc1_rb_1.pdb<br />
qsub cns.sc<br />
cd ..<br />
<br />
donedone</pre> <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; where <tt>cns.sc</tt> is a PBS macro used to submit CNS job by using PBS: <br />
<pre> #!/bin/csh<br />
#PBS -m e<br />
#PBS -q short_c<br />
#PBS -l nodes=1:GM:ppn=1<br />
#PBS -l walltime=00:56:00<br />
#PBS -o out1<br />
#PBS -j oe<br />
#PBS -N clean1<br />
cd $PBS_O_WORKDIR<br />
echo "working directory = "$PBS_O_WORKDIR<br />
set NN = `cat $PBS_NODEFILE | wc -l`<br />
echo "NN = "$NN<br />
# Run Job<br />
cns &lt; re_h2oc.inp<br />
echo "ALL Done!"</pre> <br />
&nbsp;&nbsp;&nbsp; 7.&nbsp; Run <tt>getpdb</tt> to collect refined structures. <br />
<pre> #bash<br />
for i in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20<br />
do<br />
cd ref$i<br />
cp ufc1_ref_1.pdb ../pdb/ufc1_ref_$i.pdb<br />
cp ufc1_ref_1.vio ../pdb/ufc1_ref_$i.vio<br />
cd ..<br />
done</pre> <br />
&nbsp;&nbsp;&nbsp; 8. Run script [[Media:Agrupa.txt|Agrupa]] to put all 20 conformers into a single PDB file.<tt></tt> <br />
<pre> Agrupa ufc1_ref*.pdb &gt; ufc1all.pdb</pre> <br />
=== '''Running CNS for Proteins with Metal Ions''' ===<br />
<br />
The CNS refinement for proteins with metal ions can also be performed with the new WaterrefCNS script, using the appropriate metal ion nomenclature in both the pdb coordinates and distance constraints. <br />
<br />
For manual CNS refinement please do the refinement as following: <br />
<br />
1.&nbsp; Set environment for CNS1.1 by run source /farm/users/gliu/alias.cns <br />
<pre> alias cns1 /farm/software/cns/cns_solve_1.1/intel-i686-linux_g77/bin/cns<br />
setenv CNS_TOPPAR /farm/data/gliu/cns1/</pre> <br />
In addition to the topology and parameter files, the metal ion parameter file <tt>ion.top</tt> is required. An example can be found in "/farm/users/gliu/projects/cns_cuttha_cis" with all required input files. 2.&nbsp; Prepared required files as described above (final.tbl, final cns format PDB files and put in xplorPDB dir with name as sa_#.pdb) except the PDB file should include the metal ions with format according to [http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/CNS CNS] library <tt>ion.top</tt>. cp sa_1.pdb as&nbsp; template.pdb, input files for creating mtf file Note that alignment is important. eg: <br />
<pre>ATOM 1249 OT2 ALA 83 69.296 13.232 5.744 1.00 0.00<br />
ATOM 1250 ZN+2 ZN2 150 63.086 13.789 -10.407 1.00 0.00 zinc</pre> <br />
3.&nbsp; Run generate_h2o.inp once to create temp_h2o.pdb and temp_h2o.mtf. The extra proton atom in the ligand residues, eg. HIS HD1 or CYS S, are removed by editing the generate_h2o.inp; cis proline is also defined here in the generate_h2o.inp (resid is the residue number prior the proline). <br />
<pre>{* any special prosthetic group patches can be applied here *}<br />
{===&gt;}<br />
delete select (name hg and resname cys and resid 61) end<br />
delete select (name hg and resname cys and resid 85) end<br />
delete select (name hd1 and resname his and resid 46) end<br />
delete select (name he2 and resname his and resid 83) end<br />
patch cisp<br />
reference=1=( resid 13 )<br />
end</pre> <br />
4.&nbsp; Edit generate_1.inp to remove the extra proton as did above. <br />
<br />
5.&nbsp; Run generate_20.com, this will run generate_#.inp 20 times, updating each pdb number and this creates cnsPDB/sa_cns_#.pdb <br />
<br />
6.&nbsp; Edit and run re_h2o_cu.inp, the refined pdb is kept in refinedPDB, or <br />
<br />
7.&nbsp; Use subcns to submit cns refinement by using PBS: eg, type "sh subcns". Before run subcns , make a folder " com" contains the following file. Type getpdb to get refined pdb files in refinedPDB after it finished. <br />
<br />
*cns.sc: PBS submission <br />
*cutc_h2o.mtf: mtf file created as descrive above <br />
*topology and parameter files: parallhdg5.3C.pro, parallhdg5.3.pro, topallhdg5.3.pro <br />
*re_h2o_cu.inp: input file for cns refinement<br><br />
<br />
<br> <br />
<br />
=== '''Running CNS&nbsp;with RDC Constraints''' ===<br />
<br />
Under construction<br />
<br />
=== '''Running CNS refinement on Homodimers''' ===<br />
<br />
Under construction<br><br> <br />
<br />
== '''Files for Download''' ==<br />
<br />
*[[Media:AtomtransC.tbl|atomtransC.tbl]]: atom name translation table<br />
<br />
*[[Media:Agrupa.txt|Agrupa]]: script for concatenating pdb coordinate files into one file<br />
<br />
*[[Media:Parallhdg5.3C.pro|parallhdg5.3C.pro]]: CNS parameter file<br />
<br />
*[[Media:Topallhdg5.3.pro|topallhdg5.3.pro]]: CNS topology file<br />
<br />
*[[Media:Topallhdg5.3.sol|topallhdg5.3.sol]]: CNS solvent topology file<br />
<br />
*[[Media:Parallhdg5.3.sol|parallhdg5.3.sol]]: CNS solvent parameter file<br />
<br />
*[[Media:Boxtyp20.pdb|boxtyp20.pdb]]: Water box<br />
<br />
*[[Media:Protein.link|protein.link]]: CNS peptide linkage definition file<br />
<br />
*[[Media:Generate_water.cns|generate_water.cns]]: CNS protein solvation script<br />
<br />
*[[Media:Print_coorheader.cns|print_coorheader.cns]]: CNS script<br />
<br />
*[[Media:Cns_cabm.tar.gz|cns_cabm.tar.gz]]: CABM CNS refinement protocol<br />
<br />
*[[Media:WaterRefinement_UB.tar.Z|WaterRefinement_UB.tar.Z]]: CNS water refinement package - SUNY Buffalo<br />
<br />
*[[Media:Cyana2cns.cya|cyana2cns.cya]]: CYANA script to convert coordinates and constrains to XPLOR/CNS format<br />
<br />
*[[Media:Cu_cPro_cns_example.tar.gz|Cu_cPro_cns_example.tar.gz]]: Example of CNS refinment for protein with Cu and cis-Pro<br />
<br />
*[[Media:Zn.tar|zn.tar]]: Exampleof CNS refinment for protein with zn (from Alex Lemark)<br />
<br />
*[[Media:Er317_2zn_CNSw_example.tar.gz|er317_2zn_CNSw_example.tar.gz]]: Example of CNS refinment for protein with zn (from Theresa Ramelot)<br />
<br />
== '''References''' ==<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/9757107?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=14 1.&nbsp; Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. and Warren,G.L. (1998) Crystallography &amp; NMR system: a new software suite for macromolecular structure determination. ''Acta Crystallogr. D54'', 905-921.] <br />
<br />
<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Homodimer_Structure_Calculation_with_Symmetry_Constraints&diff=3595
Homodimer Structure Calculation with Symmetry Constraints
2010-01-06T21:57:18Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
'''IMPORTANT DISCLAIMER:'''&nbsp;A number of NESG&nbsp;NMR groups are currently beta-testing sites for CYANA-3.0 and the information in this page is intended for use by the licensed members of the NESG consortium, other beta testers, and is to be used in accordance to the program licensing agreement. <br />
<br />
<br> <br />
<br />
The following page describes the setup and analysis of an automated structure determination run for symmertic homodimers starting from NOE peaklists in the framework of CYANA-3.0.<br> <br />
<br />
&nbsp;Results from CYANA&nbsp;should always be accompanied by energy refinement in CNS or NIH-XPLOR.&nbsp; Further information about the program and publication references can be found in the [http://www.cyana.org/wiki/index.php/Main_Page CYANA WIKI]&nbsp;page. <br />
<br />
<br> <br />
<br />
== <span style="font-weight: bold;">Automated Homodimer Structure Calculation Setup </span><br> ==<br />
<br />
The structure calculation with automated noesy assignments and intermolecular restraints follows the canonical CYANA&nbsp;recipe.&nbsp; Simple annealing calculations starting from a set of constraints that include RDC are easily derived by simplifying the scripts below and following the demo scripts. The program requires a sequence file (name.seq), a proton assignment list (name.prot), a noesy peaklist set (name.peaks), a CALC.cya script and an init.cya script.&nbsp;&nbsp; <br> <br />
<br />
An helpful script called '''DIMERIZE''', to be run from within CYANA, can be used to symmetrize various files such as the proton (.prot), angle and NOE constraint files:<br> <br />
<pre>!/bin/sh<br />
<br />
# Chemical shift list<br />
cp RpR324.prot RpR324d.prot<br />
awk 'NF&gt;=5 { printf("%6d%8.3f%8.3f&nbsp;%-5s%4d\n",$1+1000,$2,$3,$4,$5+200) }' $* RpR324.prot &gt;&gt; RpR324d.prot<br />
<br />
# Additional distance constraints<br />
echo "<br />
read upl D5.upl<br />
read lol D5.lol<br />
molecules symmetrize<br />
write upl D5dimer.upl<br />
write lol D5dimer.lol<br />
quit" | cyana32<br />
<br />
# angle constraints<br />
echo "<br />
read dihe.aco<br />
shift +200<br />
writeaco t.aco<br />
quit" | ancoma<br />
cat dihe.aco t.aco &gt; dihed.aco<br />
rm -f t.aco<br />
</pre> <br />
==== '''The sequence file''' ====<br />
<br />
The example sequence file, shown below in abbreviated form, now includes the doubled sequence separated by a 48-element (LL2) linker: <br> <br />
<pre>MET 1<br />
THR 2<br />
SER 3<br />
THR 4<br />
PHE 5<br />
ASP 6<br />
.<br />
.<br />
.<br />
HIS 100<br />
HIS 101<br />
<br />
PL 102<br />
LL2 103<br />
LL2 104<br />
.<br />
.<br />
LL2 148<br />
LL2 149<br />
LP 150<br />
<br />
MET 201<br />
THR 202<br />
SER 203<br />
THR 204<br />
PHE 205<br />
ASP 206<br />
.<br />
.<br />
.<br />
HIS 300<br />
HIS 301<br />
<br />
</pre> <br />
==== The CALC.cya file ====<br />
<br />
The working directory that contains all the files necessary to start the calcualtion is ready and the CALC.cya modified for the presence of RDC constraints is used:<br> <br />
<pre>peaks &nbsp;:= ali5.peaks,aro5.peaks,n3.peaks,xfilt.peaks # names of NOESY peak lists<br />
prot &nbsp;:= RpR324d.prot # names of chemical shift lists<br />
restraints &nbsp;:= ssa.cya,hbonds.upl,hbonds.lol,manual.upl,dihed.aco # additional (non-NOE) constraints<br />
tolerance &nbsp;:= 0.04,0.025,0.3 # chemical shift tolerances: HX2-HX1-X1<br />
calibration&nbsp;:= # NOE calibration parameters<br />
structures &nbsp;:= 100,20 # number of initial, final structures<br />
steps &nbsp;:= 10000 # number of torsion angle dynamics steps<br />
rmsdrange &nbsp;:= 10..80 # residue range for RMSD calculation<br />
randomseed &nbsp;:= 56231 # random number generator seed<br />
<br />
ssa<br />
noeassign peaks=$peaks prot=$prot autoaco<br />
</pre> <br />
==== Defining inter-chain only peaks ====<br />
<br />
Notice the peak contain a list of intermolecular NOEs called xfilt.peaks.&nbsp; It is possible to instruct the program to make only interchain NOE&nbsp;assignments for the peaklist by using the 9 U&nbsp;xeasy color-code as shown below, the remaining peaklists can be treated as usual as a mixture of inter and intra chain NOEs.<br> <br />
<pre># Number of dimensions 3<br />
#FORMAT xeasy3D<br />
#INAME 1 h<br />
#INAME 2 C<br />
#INAME 3 H<br />
#CYANAFORMAT hCH<br />
1 8.072 35.125 4.110 9 U 1.000E+06 0.000E+00 e 0 0 0 0<br />
2 2.584 33.247 4.446 9 U 3.610E+05 0.000E+00 e 0 0 0 0<br />
3 6.801 42.287 3.199 9 U 4.280E+05 0.000E+00 e 0 0 0 0<br />
4 6.799 42.276 2.940 9 U 3.440E+05 0.000E+00 e 0 0 0 0 <br />
<br />
</pre> <br />
==== The Init.cya file <br> ====<br />
<br />
The new intstructions for symmetric dimer treatment are included in the init.cya file, in the example below molecule indentity and molecule symmetry are parametrized and adjustable (probably the parameters have been optimized so results tweaking the weight_ide and weight_sym should be carefully). <br />
<pre>name:=RpR324<br />
rmsdrange:=10-80<br />
cyanalib<br />
nproc:=8<br />
read seq $name.seq<br />
molecules define 1..101 201..301<br />
molecule identity<br />
weight_ide=0.03<br />
molecule symdist "CA 1..101" "CA 201..301"<br />
weight_sym=0.0025<br />
<br />
</pre> <br />
The weight of the symmetry and indentity constraints does not appear to be parametrized in the target function as a penalty.&nbsp; Further explanation will hopefully become available with the official release. <br />
<br />
The use of MPI&nbsp;routine is highly recommended due to the length of the calculation. <br />
<br />
<br> <br />
<br />
== <span style="font-weight: bold;">Output analysis </span><br> ==<br />
<br />
The output analysis is carried out in the usual manner as for CYANA -2.1. <br> <br> <br> <br />
<br />
<br> <br />
<br />
<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Structure_Calculation_With_RDC%27s_Using_CYANA&diff=3594
Structure Calculation With RDC's Using CYANA
2010-01-06T21:56:51Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
'''IMPORTANT DISCLAIMER:'''&nbsp;A number of NESG&nbsp;NMR groups are currently beta-testing sites for CYANA-3.0 and the information in this page is intended for use by the licensed members of the NESG consortium, other beta testers, and is to be used in accordance to the program licensing agreement. <br />
<br />
<br> <br />
<br />
The following page describes the setup of and analysis of an automated structure determination starting from NOEs peaklists and residual dipolar coupling (RDC) constraints in the framework of CYANA 3.0.<br> <br />
<br />
CYANA version 3.0 incorporates many new features including:<br> <br />
<br />
a) inclusion of residual dipolar coupling in structure calculation (RDC) <br> <br />
<br />
b) inclusion of pseudocontact shifts from paramagnetic centers in structure calculation (PCS)<br> <br />
<br />
c) improved function for symmetric dimers and annotation of intermolecular NOE&nbsp;contacts in the peaklist using the xeasy color code notation. &nbsp;<br> <br />
<br />
Good agreement between global orientational constraints from RDC&nbsp;and distance information from NOEs is very important to achieve better structures by NMR.&nbsp; Results should always be accompanied by energy refinement in CNS or NIH-XPLOR.&nbsp; Further information about the program and publication references can be found in the [http://www.cyana.org/wiki/index.php/Main_Page CYANA WIKI]&nbsp;page.<br> <br />
<br />
<br> <br />
<br />
== <span style="font-weight: bold;">Automated NOE and RDC and Structure Calculation Setup </span><br> ==<br />
<br />
The structure calculation with automated noesy assignments and RDC&nbsp;restraints follows the canonical CYANA&nbsp;recipe.&nbsp; Simple annealing runs starting from a set of constraints that include RDC are easily derived by simplifying the scripts below and following the demo scripts. The program requires a sequence file (name.seq), a proton assignment list (name.prot), a noesy peaklist set (name.peaks), an RDC&nbsp;list (name.rdc), a CALC.cya script and an init.cya script.&nbsp;&nbsp; <br> <br />
<br />
==== Sequence file<br> ====<br />
<br />
The sequence file now includes the RDC&nbsp;tensor origin separated by dummy linker residues (LL5): <br> <br />
<pre>MET 1<br />
THR 2<br />
SER 3<br />
THR 4<br />
PHE 5<br />
ASP 6<br />
ARG 7<br />
VAL 8<br />
ALA 9<br />
THR 10<br />
<br />
PL 350<br />
LL5 351<br />
LL5 352<br />
LL5 353<br />
LL5 354<br />
LL5 355<br />
ORI 360<br />
<br />
</pre> <br />
<br> <br />
<br />
==== RDC&nbsp;constraint file<br> ====<br />
<br />
The RDC&nbsp;list supports multiple interatomic vectors in multiple media. RDC&nbsp;with distinct scaling factors and distinct ORI&nbsp;residue numbers are listed in a single file. The program supports the Da (magnitude) and R (Rhombicity) notation typical of programs such as PALES, REDCAT etc. Below is a sample RDC file that includes N-H, N-CA (intra), and N-C' (sequential) vectors in one medium with adequate error (here the following errors were used:&nbsp; ~10&nbsp;% the RDC spread for N-H vectors, the error determined by analysis of ''J''-modulated experiments for N-CA and N-C' RDC measurement) and <span style="text-decoration: underline;">[http://www.ncbi.nlm.nih.gov/pubmed/18388951 weight factors]:</span><br> <br />
<pre># Orientation Magnitude Rhombicity ORI residue number<br />
1 5.39535 0.63125 360<br />
# First atom Second atom RDC Error Weight Orientation <br />
7 ARG H 7 ARG N 5.936 2.000 1.000 1<br />
8 VAL H 8 VAL N 3.827 2.000 1.000 1<br />
9 ALA H 9 ALA N -2.822 2.000 1.000 1<br />
10 THR H 10 THR N -0.674 2.000 1.000 1<br />
11 ILE H 11 ILE N 4.945 2.000 1.000 1<br />
12 ILE H 12 ILE N 1.709 2.000 1.000 1<br />
13 ALA H 13 ALA N -1.336 2.000 1.000 1<br />
#<br />
4 THR N 3 SER C 1.267 0.095 8.330 1<br />
5 PHE N 4 THR C -0.246 0.207 8.330 1 <br />
7 ARG N 6 ASP C 0.161 0.052 8.330 1 <br />
8 VAL N 7 ARG C 0.439 0.034 8.330 1 <br />
9 ALA N 8 VAL C -0.076 0.040 8.330 1 <br />
10 THR N 9 ALA C -0.957 0.048 8.330 1 <br />
11 ILE N 10 THR C 1.123 0.022 8.330 1 <br />
12 ILE N 11 ILE C -0.440 0.037 8.330 1 <br />
13 ALA N 12 ILE C 0.065 0.026 8.330 1 <br />
#<br />
3 SER N 3 SER CA 0.251 0.199 8.330 1<br />
4 THR N 4 THR CA -0.265 0.258 8.330 1<br />
5 PHE N 5 PHE CA -0.499 0.281 8.330 1<br />
7 ARG N 7 ARG CA -0.457 0.200 8.330 1<br />
8 VAL N 8 VAL CA -0.481 0.154 8.330 1<br />
9 ALA N 9 ALA CA 0.349 0.083 8.330 1<br />
10 THR N 10 THR CA 0.548 0.121 8.330 1<br />
11 ILE N 11 ILE CA -0.091 0.111 8.330 1<br />
12 ILE N 12 ILE CA -0.678 0.078 8.330 1<br />
13 ALA N 13 ALA CA 1.014 0.108 8.330 1</pre> <br />
Multiple media (e.g. orientations) should be listed as follows: <br> <br />
<pre># Orientation Magnitude Rhombicity ORI residue number<br />
1 5.39535 0.63125 360<br />
2 7.55656 0.58200 370<br />
</pre> <br />
and the sequence should be modified to include further links and ORI:<br> <br />
<pre>ALA 9<br />
THR 10<br />
<br />
PL 350<br />
LL5 351<br />
LL5 352<br />
LL5 353<br />
LL5 354<br />
LL5 355<br />
ORI 360<br />
LL5 361<br />
LL5 362<br />
LL5 363<br />
LL5 364<br />
LL5 365<br />
ORI 370</pre> <br />
<br> <br />
<br />
==== Initial model-free determination of Da and R from assigned RDCs ====<br />
<br />
If the values of Da and R are not known they can be determined using any desired software OR&nbsp;using the FindTensor.cya script below. The program yields results that are equivalent to PALES assiuming the same fitting method is employed.<br> <br />
<pre>## 8DEMOS: FindTensor - Determine alignment tensor<br />
##<br />
## Determine magnitude and rhombicity of the alignment tensor<br />
## from input RDCs<br />
<br />
<br />
# determine tensor from histogram, no structure needed<br />
<br />
read rdc phage_all_mono.rdc<br />
print "&nbsp;&nbsp;&nbsp; Input alignment tensor:"<br />
do i 1 orientations<br />
&nbsp; print "&nbsp;&nbsp;&nbsp; Orientation $i: magnitude = $magnitude(i) Hz, rhombicity = $rhombicity(i)."<br />
end do<br />
<br />
rdc fittensor method=simplex&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; # (can take several minutes)<br />
#rdc fittensor method=gridsearch&nbsp;&nbsp; # systematic search (very slow)<br />
<br />
<br />
# alternatively, determine tensor from given structure by SVD<br />
<br />
read rdc phage_all_mono.rdc<br />
read pdb final.pdb<br />
overview<br />
<br />
</pre> <br />
The dummy values in the rdc list are read initially and they can be updated after running the FindTensor.cya routine. If no models are present (e.g. final.pdb) the program will terminate with a warning. <br> <br />
<br />
<br> <br />
<br />
==== The CALC.cya file ====<br />
<br />
The working directory that contains all the files necessary to start the calcualtion is ready and the CALC.cya modified for the presence of RDC constraints is used:<br> <br />
<pre>peaks &nbsp;:= ali5.peaks,aro5.peaks,n3.peaks # names of NOESY peak lists<br />
prot &nbsp;:= RpR324.prot # names of chemical shift lists<br />
restraints &nbsp;:= ssa.cya,phage_all_mono.rdc # additional (non-NOE) constraints<br />
tolerance &nbsp;:= 0.04,0.025,0.3 # chemical shift tolerances: HX2-HX1-X1<br />
calibration&nbsp;:= # NOE calibration parameters<br />
structures &nbsp;:= 100,20 # number of initial, final structures<br />
steps &nbsp;:= 10000 # number of torsion angle dynamics steps<br />
rmsdrange &nbsp;:= 10..80 # residue range for RMSD calculation<br />
randomseed &nbsp;:= 56231 # random number generator seed<br />
<br />
weight_rdc = 0.02 # weight for RDC restraints<br />
cut_rdc = 0.2 # cutoff for RDC violation output<br />
<br />
ssa<br />
noeassign peaks=$peaks prot=$prot autoaco<br />
<br />
</pre> <br />
Notice that the 'restraints' row contain the forced stereospecifically assigned methyls and sidechain NH's and the .rdc file.&nbsp; Also, the NOE vs. RDC&nbsp;weight is set by the weight_rdc and cut_rdc functions.&nbsp; The remaining instructions are identical to the CYANA-2.1 file.&nbsp; <br />
<br />
<br> <br />
<br />
==== The init.cya file ====<br />
<br />
Further parameters are specified in the init.cya file below:<br> <br />
<pre>name:=RpR324<br />
rmsdrange:=10-80<br />
cyanalib<br />
nproc:=8<br />
read seq $name.seq<br />
rdcdistances</pre> <br />
The above script is intended to run off a single dual-quad core machine (nproc=8).&nbsp; <br />
<br />
===== rdcdistances.cya file =====<br />
<br />
Please note the rdcdistance.cya macro is being called by the init.cya setup file.&nbsp; This file, located in the cyana-3.0/macro directory contains the supported RDC&nbsp;vectors, more vectors could potentially be added such as Trp N<sup>ε1</sup>-H<sup>ε1</sup> that maybe useful in deuterated sample to direct the large hydrophobic sidechain.&nbsp; <br> <br />
<pre># Copyright (c) 2002-08 Peter Guntert. All rights reserved.<br />
## 7MACROS: rdcdistances - CYANA macro<br />
##<br />
## Parameters: (none)<br />
##<br />
# dipole definition format: atom1_name atom2_name atom1_index atom2_index<br />
# if indexes are missing, zeros are assumed<br />
<br />
var info echo<br />
<br />
syntax<br />
<br />
info:=none; echo:=off<br />
rdc distance "N&nbsp; H"&nbsp;&nbsp;&nbsp; distance=1.041<br />
rdc distance "CA HA"&nbsp;&nbsp; distance=1.117<br />
rdc distance "C&nbsp; CA"&nbsp;&nbsp; distance=1.525<br />
rdc distance "C&nbsp; N"&nbsp;&nbsp;&nbsp; distance=2.461<br />
rdc distance "C&nbsp; N -1" distance=1.329<br />
rdc distance "CA N"&nbsp;&nbsp;&nbsp; distance=1.458<br />
rdc distance "CA N -1" distance=2.425<br />
rdc distance "CA H"&nbsp;&nbsp;&nbsp; distance=2.117<br />
rdc distance "CA H -1" distance=2.533<br />
rdc distance "C&nbsp; H -1" distance=2.000<br />
rdc distance "C&nbsp; HA"&nbsp;&nbsp; distance=2.144<br />
rdc distance "CB HB"&nbsp;&nbsp; distance=1.080<br />
rdc distance "CA CB"&nbsp;&nbsp; distance=1.532<br />
unset info<br />
print "&nbsp;&nbsp;&nbsp; Standard RDC distances defined."<br />
</pre> <br />
<br> <br />
<br />
==== Notes on run execution ====<br />
<br />
The command line execution form single machine or cluster using MPI implememntation (highly recommended) is carried on as usual:<br> <br />
<pre>/cyana-3.0/cyana CALC &gt; &amp; log &amp;<br />
</pre> <br />
the MPI&nbsp;is launched using the script called, for example, submit_cyana:<br> <br />
<pre>#!/bin/bash<br />
#PBS -S /bin/bash<br />
#PBS -N cyana<br />
#PBS -lnodes=6:ppn=8<br />
lamboot ~/bhost.def<br />
cd /farm/users/prossi/RpR324_structure/cyana_new_mono2<br />
/opt/openmpi/tcp-gnu/bin/mpirun /farm/software/cyana-3.0-mpi/cyana CALC.cya<br />
lamhalt</pre> <br />
<br>with the command:<br> <br />
<pre>qsub -q @master3 submit_cyana<br />
</pre> <br />
The starting scripts are highly system specific they are almost guaranteed NOT&nbsp;to work on your sytem and are given here for general information only. <br> <br />
<br />
<br> <br />
<br />
== <span style="font-weight: bold;">Output analysis </span><br> ==<br />
<br />
The output analysis is carried out in the usual manner, it should be noted that, during the calculation the specified values Da and R are kept fixed.&nbsp; Following the final cycle a new model-based estimate of Da and R&nbsp;is calculated and used to compute the RDC violations and their contribution to the target function and the quality factor (Q). The resulting target function will be increased by the number and extent of RDC&nbsp;violations in addition to other violations from dihedral, vdw, and NOEs restraints. <br />
<br />
A partial output file is given below (final.ovw):<br> <br />
<pre> <br />
Structural statistics:<br />
<br />
str target upper limits van der Waals RDCs<br />
function # rms max # sum max # rms max<br />
1 8.98 5 0.0089 0.40 12 18.5 0.37 13 0.2508 2.27<br />
2 8.96 7 0.0083 0.31 15 17.4 0.38 19 0.2656 2.27<br />
3 9.69 11 0.0122 0.54 15 18.6 0.38 18 0.2595 2.28<br />
4 9.63 6 0.0068 0.20 16 18.9 0.38 18 0.2604 2.27<br />
5 9.41 14 0.0089 0.22 17 20.4 0.37 14 0.2521 2.26<br />
6 9.80 10 0.0085 0.24 17 19.7 0.38 15 0.2563 2.27<br />
7 10.52 12 0.0158 0.78 15 19.1 0.38 13 0.2554 2.29<br />
8 10.05 10 0.0084 0.18 18 20.3 0.42 18 0.2587 2.28<br />
9 10.57 7 0.0079 0.29 19 18.3 0.53 16 0.3169 2.43<br />
10 10.38 13 0.0088 0.21 22 20.5 0.37 21 0.2636 2.28<br />
11 10.33 7 0.0067 0.16 17 19.6 0.64 15 0.2663 2.28<br />
12 9.93 9 0.0090 0.29 19 20.6 0.38 20 0.2541 2.27<br />
13 10.12 12 0.0098 0.23 21 19.9 0.47 13 0.2681 2.30<br />
14 10.53 7 0.0077 0.19 23 19.3 0.38 11 0.3145 2.40<br />
15 10.96 8 0.0089 0.31 21 22.6 0.38 12 0.2639 2.28<br />
16 10.50 12 0.0100 0.29 23 20.1 0.37 15 0.2696 2.27<br />
17 10.56 14 0.0119 0.30 23 21.1 0.37 16 0.2736 2.33<br />
18 10.75 17 0.0146 0.60 21 20.0 0.37 12 0.2547 2.27<br />
19 10.88 18 0.0125 0.38 19 23.3 0.38 10 0.2506 2.30<br />
20 10.88 8 0.0083 0.26 23 21.4 0.42 15 0.2644 2.28<br />
<br />
Ave 10.17 10 0.0097 0.32 19 20.0 0.41 15 0.2660 2.29<br />
+/- 0.59 4 0.0024 0.15 3 1.4 0.07 3 0.0177 0.04<br />
Min 8.96 5 0.0067 0.16 12 17.4 0.37 10 0.2506 2.26<br />
Max 10.96 18 0.0158 0.78 23 23.3 0.64 21 0.3169 2.43<br />
Cut 0.10 0.20 0.20<br />
<br />
Constraints violated in 6 or more structures:<br />
# mean max. 1 5 10 15 20<br />
Upper HA PRO 19 - HB3 ARG 20 5.50 15 0.11 0.21 +++++++ ++++ ++*+ peak 970<br />
Upper HA ILE 23 - QB SER 27 5.34 6 0.09 0.16 + ++ + +* peak 276<br />
Upper HA ILE 30 - HB2 LEU 33 4.95 10 0.10 0.15 ++++ ++ ++ +* peak 313<br />
VdW CB ALA 69 - H THR 70 2.55 18 0.24 0.28 ++++++++ ++++ *+++++<br />
VdW O THR 71 - N PHE 75 2.75 12 0.20 0.32 ++* +++ + ++ +++<br />
VdW O PHE 75 - C VAL 76 2.80 13 0.21 0.31 ++++ + *+++++++<br />
VdW CG1 VAL 76 - HG2 LYS 78 2.60 9 0.17 0.25 ++* +++++ +<br />
VdW HG3 LYS 78 - C LYS 78 2.50 14 0.22 0.36 +++*++ +++++ + + +<br />
Ori 1 N ALA 69 - CA ALA 69 -2.84 20 1.95 2.04 +++++++++++++*++++++<br />
Ori 1 H ALA 69 - N ALA 69 -3.32 20 2.01 2.33 ++++++++++++++++*+++<br />
Ori 1 N THR 70 - C ALA 69 0.25 16 0.31 1.06 ++++++++*+++ + + ++<br />
Ori 1 H ASN 79 - N ASN 79 -4.71 10 0.17 0.32 + *+ + ++++ + +<br />
Ori 1 H GLY 92 - N GLY 92 -5.54 8 0.15 0.51 ++++* + ++<br />
Ori 1 H LEU 94 - N LEU 94 0.42 6 0.14 0.54 +*+ +++<br />
3 violated distance restraints.<br />
5 violated van der Waals restraints.<br />
6 violated residual dipolar coupling restraints.<br />
<br />
<br />
RDC statistics:<br />
Correlation coefficient &nbsp;: 0.906 +/- 0.003 (0.899..0.909, best in conformer 4)<br />
Q = rms(Dcalc-Dobs)/rms(Dobs): 42.709 +/- 0.583&nbsp;% (42.073..44.194)<br />
Q normalized by tensor &nbsp;: 32.943 +/- 0.638&nbsp;% (32.231..34.776)<br />
Alignment tensor magnitude &nbsp;: 5.881 +/- 0.045 Hz (5.760..5.950, best 5.898; input 5.898)<br />
Alignment tensor rhombicity &nbsp;: 0.537 +/- 0.007 (0.524..0.556, best 0.539; input 0.539)<br />
<br />
<br />
</pre> <br />
<br> <br />
<br />
<br> <br />
<br />
<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Homodimer_Structure_Calculation_Using_CYANA&diff=3593
Homodimer Structure Calculation Using CYANA
2010-01-06T21:56:07Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
CYANA versions 2.1 and up can perform structure calculation with automated NOE assignment for symmetric homodimers. The algorithm is slightly different from what is used to calculate monomers or asymmetric oligomers. Additional restraints are introduced to keep the molecule and the force field symmetric.&nbsp; A tutorial for homodimer structure calculation using CYANA 3.0 is avaliable [http://www.cyana.org/wiki/index.php/Homodimer_structure_calculation_with_automated_NOESY_assignment on-line]. <br />
<br />
== '''Input Data''' ==<br />
<br />
Chemical shift assignment is performed for a single protein chain. Thus, the input for a symmetric homodimer calculation must include: <br />
<br />
*duplicated sequence file <br />
*duplicated atom list <br />
*duplicated external constraint files, if any<br />
<br />
=== '''Duplicated Sequence File''' ===<br />
<br />
The protein sequence must be repeated twice with a linker between protein chains as in this example:<br> <br />
<pre>MET 11<br />
ALA 12<br />
GLU 13<br />
VAL 14<br />
GLU 15<br />
...<br />
GLU 106<br />
PL 107<br />
LL2 108<br />
...<br />
LL2 128<br />
LL2 129<br />
LP 130<br />
MET 211<br />
ALA 212<br />
GLU 213<br />
VAL 214<br />
GLU 215<br />
...<br />
GLU 306</pre> <br />
The CYANA residue library provides the following linker pseudo-residues: <tt>PL</tt>, <tt>LP</tt>, <tt>LL</tt>, <tt>LL2</tt> and <tt>LL5</tt>. The first two are special transition "residues", which are required to start and terminate the linker, respectively. The last three are intermediate linker "residues" of differing sizes, with "bonds" of standard length, double length and quintuple length. The "atoms" of these linker "residues" have zero mass and zero Van-der-Vaals radii, thus the linker can freely pass through the structure during simulated annealing. The "bonds" of the linker cannot be stretched, however, and it is necessary to use a linker of sufficient length to avoid putting unnatural strain on the subunits. <br />
<br />
It is convenient to have the residue numbers of the second subunit shifted by 100 or 200 compared to the first subunit. The first subunit in this example protein has residue numbers 11 to 106, while the second subunit has residue numbers from 211 to 306. It is not necessary to insert a linker exactly 94 residues long - hops in the residue numbers, such as between <tt>LP 130</tt> and <tt>MET 211</tt>, are accepted. <br />
<br />
=== '''Duplicated Atom List''' ===<br />
<br />
Chemical shifts in the atom list (.prot file) should be duplicated to include assignments for the second chain. <br />
<pre> 155 119.362 0.000 N 11<br />
156 8.323 0.004 HN 11<br />
157 56.722 0.000 CA 11<br />
158 4.318 0.005 HA 11<br />
159 31.426 0.000 CB 11<br />
160 2.010 0.000 HB2 11<br />
161 2.137 0.000 HB3 11<br />
...<br />
1818 119.362 0.000 N 211<br />
1819 8.323 0.004 HN 211<br />
1820 56.722 0.000 CA 211<br />
1821 4.318 0.005 HA 211<br />
1822 31.426 0.000 CB 211<br />
1823 2.010 0.000 HB2 211<br />
1824 2.137 0.000 HB3 211</pre> <br />
=== '''Duplicated External Constraints''' ===<br />
<br />
All external constraints, such as intramolecular UPLs, ACOs and hydrogen bond constraints, must be duplicated to include both chains, otherwise the symmetry of the model will be broken. Intermolecular UPLs should also be given as symmetric pairs. <br />
<br />
=== '''Modified init.cya''' ===<br />
<br />
It is necessary to add a <tt>molecules define</tt> statement to the <tt>init.cya</tt> file. The statement tells CYANA to perform the symmetric homodimer calculation, and the residue ranges are used to maintain symmetry.<br> <br />
<pre>name:=hr2106-cyana<br />
cyanalib<br />
read seq $name.seq<br />
rmsdrange:=12..104,212..304<br />
molecules define 11..106 211..306 </pre> <br />
== '''Automatic Calculation''' ==<br />
<br />
Run the same <tt>CALC.cya</tt> macro as usual. <br />
<br />
Though in principle CYANA is able to perform calculation of homodimer structures without external constraints, the proper convergence has not been observed yet, at least in Szyperski's lab. The typical result is two separate protein chains, instead of a dimer structure. The poor convergence is not that surprising given that symmetric homodimer calculation and NOE assignment is a massively degenerate problem, compared to the monomer case. <br />
<br />
The standard approach of using external intraresidue- and short-range UPL together with ACOs from TALOS does not improve convergence, since these constraints are purely ''intramolecular''. To "nudge" the structure calculation in the proper direction external ''intermolecular'' UPLs should be supplied. These are obtained from an X-filtered NOESY experiment on a 50:50 labeled/unlabeled protein mixture. <br />
<br />
It is not necessary to generate constraints for every cross-peak in the X-filtered NOESY spectrum. Instead, one should focus on a few strong cross-peaks, which can be '''manually''' assigned with absolute certainty. For example, the structure calculation of StR106 converged to the correct dimer geometry after adding a single intermolecular UPL constraint between an aromatic ring proton and a methyl group. <br />
<br />
== '''Manual Calculation''' ==<br />
<br />
*Manual run<br />
<br />
Similar to the automatic run, modify the <tt>seq</tt> file, <tt>prot</tt> list, <tt>init.cya</tt>, and run the CYANA manual CALC macro as usual.</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Automated_NOESY_Assignment_Using_CYANA&diff=3592
Automated NOESY Assignment Using CYANA
2010-01-06T21:54:57Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
Below is the description of how to run CYANA 2.1 for automated NOE assignment if you are working with CARA.&nbsp; A tutorial for performing structure calculations with automated NOESY assignments using CYANA 3.0 is available [http://www.cyana.org/wiki/index.php/Structure_calculation_with_automated_NOESY_assignment on-line]. <br />
<br />
== '''Input files''' ==<br />
<br />
Required files <br />
<br />
*Initialization file <tt>init.cya</tt>. <br />
*SequenceList in XEASY format - usually <tt>XXXX.seq</tt>, where XXXX is the NESG ID. <br />
*AtomList in XEASY format <tt>XXXX.prot</tt> . Chemical shifts should be real, not folded. Make sure that you are using the most recent file. Atom labels should be swapped if using stereospecific assignments. <br />
*Separate unfolded PeakList for <sup>15</sup>N and <sup>13</sup>C NOESY: <tt>n.peaks</tt>, <tt>ali.peaks</tt>, <tt>aro.peaks</tt>.<br />
<br />
Optional files <br />
<br />
*Stereospecific assignment script (such as <tt>stereofound.cya</tt> from FOUND/HABAS). Note that this script should contain only <tt>atom stereo</tt> declarations, but no <tt>atom swap</tt> statements! Atom labels must be already swapped in the AtomList and external UPL files. <br />
*External UPL files, such as <tt>short.upl</tt>. Atom labels should be swapped if using stereospecific assignments. <br />
*External ACO files, such as <tt>gridsearch.aco</tt> output of FOUND/HABAS.<br />
<br />
== '''Format Conversion''' ==<br />
<br />
The input files (sequence, atom list, ACOs and UPLs) must adhere to the IUPAC nomenclature used by CYANA 2.1 (i.e., <tt>H instead of HN</tt>, etc.). CARA is fully compatible with this nomenclature, while data from other programs may need to be converted. <br />
<br />
=== '''Conversion from XEASY/DYANA/CYANA 1.X''' ===<br />
<br />
For the automated / <tt>noesyassign</tt> runs of CYANA, please make sure that your chemical shift list conforms to the IUPAC nomenclature (i.e., <tt>H instead of HN</tt>). To update your atom names, do the following in CYANA: <br />
<pre>translate dyana<br />
read protein.prot<br />
translate off<br />
write protein-cyana.prot</pre> <br />
The <tt>protein-cyana.prot</tt> file now contains all of the correct atom names for CYANA. <br />
<br />
You may need to do the same with UPLs created in DYANA or CYANA 1.X <br />
<br />
See the <tt>~/demo/details/MigrateFromDyanaCyana1.cya</tt> example script in the CYANA 2.1 installation directory for details. <br />
<br />
=== '''Conversion from Sparky''' ===<br />
<br />
CYANA can also read BMRB format chemical shift by using following commands: <br> <br />
<pre>...<br />
read bmrb protein.bmrb<br />
write prot protein.prot </pre> <br />
For Sparky users, please use Sparky command <tt>xe</tt> to write out XEASY format peaklists. <br />
<br />
=== '''Splitting the simultaneous NOESY peaklist''' ===<br />
<br />
When working with CARA it is not necessary to provide external ACO and UPL files. In CARA spin assignments are not derived from peak lists, and there is less impact from CYANA modifying existing peaks assignments. When external constraints are employed there are usually fewer peaks assigned and fewer UPLs derived. Thus it is recommended to use external UPL and ACO files only if there are convergence problems without them. <br />
<br />
When using a simultaneous 3D NOESY peaklsit XEASY, you need to generate separate peaklists with UBNMR. The following UBNMR macro is provided as an example. It calculates proper <sup>15</sup>N chemical shifts and peak positions, and writes out separate <tt>nnoe.peaks</tt> and <tt>cnoe.peaks</tt> peaklists. Modify the numbers to reflect the proper <sup>15</sup>N and <sup>13</sup>C carrier offsets (in ppm) and the spectral width ratios (<tt>sw2/sw2N</tt>).<br> <br />
<pre>init<br />
read seq xxx.seq<br />
write seq xxxseq.bmrb autoBMRB<br />
read prot xxx-simnoesy.prot<br />
read peaks xxx-simnoesy.peaks<br />
update peak shift N -35.700 1.0822510<br />
update peak shift N 117.273 1<br />
write peaks ncnoe.peaks<br />
split ncnoe.peaks nnoe.peaks cnoe.peaks<br />
update proton shift N -35.700 1.0822510<br />
update proton shift ND2 -35.700 1.0822510<br />
update proton shift NE -35.700 1.0822510<br />
update proton shift NE2 -35.700 1.0822510<br />
update proton shift NE1 -35.700 1.0822510<br />
update proton shift N 117.273 1<br />
update proton shift ND2 117.273 1<br />
update proton shift NE 117.273 1<br />
update proton shift NE2 117.273 1<br />
update proton shift NE1 117.273 1<br />
write prot noe.prot</pre> <br />
=== '''External UPL Files''' ===<br />
<br />
<tt>noeassign</tt> employs so-called "sum of r<sup>-6</sup>" averaging method (<tt>peaks calibrate</tt>) to calibrate peaklists and interpret UPLs during calculation. Therefore, external UPLs should ideally be calibrated with the same method. <br />
<br />
If you supply UPL constraints created with CALIBA (CALIBA uses "center" averaging), you should be aware that these constraints will be too loose. <br />
<br />
=== '''Using Unassigned Peaklists''' ===<br />
<br />
If you are using completely unassigned peaklist (for example, picke from scratch in CARA), then you will need to add the following line to the peaklist header: <br />
<pre>#CYANAFORMAT HNh</pre> <br />
or <br />
<pre>#CYANAFORMAT HCh</pre> <br />
The lowercase h denotes the indirect (NOE) <sup>1</sup>H dimension. <br />
<br />
If your peaklist contains assigned peaks, then CYANA will be able to determine the peaklist dimensions based on these assignments. <br />
<br />
== '''Running Automated Structure Calculation with CYANA 2.1''' ==<br />
<br />
#Create a working subdirectory (for example, <tt>structure/cyana21/calc1</tt>). <br />
#Create an init.cya file as described in [[CYANA|Getting Started or]] copy a previously used file. Set an appropriate RMSD calculation range. <br />
#Copy the latest sequence (<tt>XXXX.seq</tt>) and peaklist files (<tt>n.peaks</tt>, <tt>ali.peaks</tt> and <tt>aro.peaks</tt>) into the working directory. The sequence file and peaklist should in principle be the same as those used to [[FOUND|run FOUND]]. <br />
#Copy the updated atomlist (<tt>XXXX.prot</tt>). The spin labels in it should be swapped according to the [[FOUND|output of FOUND]]. <br />
#If you used FOUND, then copy the <tt>gridsearch.aco</tt> file from the previous FOUND run. <br />
#If you used FOUND, then copy the <tt>stereofound.cya</tt> file from the previous FOUND run. Make sure that incorrect stereospecific assignments have been commented out or removed. <br />
#(Optional) Generate the short-range UPL (<tt>short.upl</tt>) file based on the existing peak assignments. This is more convenient to do on a workstation. You can use the [[Media:Make_short.cya|make_short.cya]] script (see below). Alternatively, you can define a <tt>KEEP</tt> subroutine in the <tt>CALC.cya</tt> file. <br />
#Download the [[Media:CALC_noeassign.cya|CALC.cya]] script (see below) and modify it according to the input data.<br />
<br />
You can choose whether you want to run structure calculation on a local Linux workstation or on the U2 Linux cluster. The typical machine times on a single workstation are 1.5 - 3 hours, depending on the protein size. Calculations on the cluster take only 15-30 minutes, but there my be additional queue waiting time. On weekdays during working hours (9 a.m. - 4 p.m.) there are 10 dual-processor nodes reserved for us only, and there is no waiting time. <br />
<br />
Check the [http://www.ccr.buffalo.edu/hotpages/content/u2/queue.htm queue status page] and the [http://www.ccr.buffalo.edu/hotpages/content/u2/nodes.htm nodemap page] to see the current system loads on U2. <br />
<br />
To run calculations on the U2 Linux cluster: <br />
<br />
#Log in to <tt>u2.ccr.buffalo.edu</tt> <br />
#Change directory to <tt>/san/projects1/szypersk/</tt>. <br />
#Create a working subdirectory (like <tt>username/XXXX/cyana21</tt>) <br />
#Copy the entire subdirectory <tt>calc1</tt>. You can use <tt>gftp</tt>, <tt>scp</tt> or <tt>sftp</tt>. <br />
#Download the PBS submission script [[Media:Cyana.pbs|cyana.pbs]] (see below). Modify it if needed. <br />
#Type <tt>qsub cyana.pbs</tt> to submit you job.<br />
<br />
To run calculations on a workstation: <br />
<br />
#Start CYANA 2.1 by typing <tt>cyana21</tt> <br />
#Enter <tt>CALC</tt> at the cyana prompt.<br />
<br />
<br> <br />
<br />
== '''Output files''' ==<br />
<br />
*<tt>final.pdb</tt> - resulting structure <br />
*<tt>final.ovw</tt> - final overview file <br />
*<tt>final.upl</tt> - final UPL file (unambiguous constraints; atom labels may be swapped) <br />
*<tt>*-final.prot</tt> - final atom list (chemical shifts unchanged?; atom labels may be swapped) <br />
*<tt>finalstereo.cya</tt> - stereospecific assignment file (to find swapped atom pairs see calculation log) <br />
*<tt>*-cycle7.peaks</tt> - assigned peaklists (in CYANA 2.1 format with multiple assignments) <br />
*<tt>cycleX.*</tt> - UPL, OVW, PDB and NOA files for cycle X (ambiguous constraints in UPL files)<br />
<br />
Macro <tt>noeassign</tt> in CYANA 2.1 performs 7 routine calculation cycles and one final cycle. The output files are labeled <tt>cycle1.*</tt>, <tt>cycle2.*</tt> ... <tt>cycle7.*</tt> and <tt>final.*</tt> with appropriate extensions. Additional stereospecific assignment search is performed after cycle 7, therefore files, <tt>final.upl</tt> and <tt>*-final.prot</tt> likely have some labels swapped. <br />
<br />
Assigned peak lists are saved after cycle 7. They may have multiple assignments for some peaks thus not being fully compatible with XEASY. <br />
<br />
Always check the output of CYANA calculation for the results of <tt>peakcheck</tt> command. It is executed before the first calculation cycle and reports various inconsistencies in the atom list and peak lists. In the end, many UPL violations can be traced back to mistakes in assignment or mis-picked peaks. <br />
<br />
== '''Example scripts''' ==<br />
<br />
Below are the key scripts for running CYANA. See the demo subdirectory of CYANA installation for more details. <br />
<br />
=== '''make_short.cya''' ===<br />
<pre>peaks &nbsp;:= n,ali,aro # names of peak lists<br />
prot &nbsp;:= $name # names of proton lists<br />
tolerance &nbsp;:= 0.05,0.02,0.3 # chemical shift tolerances<br />
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)<br />
calibration:= 1.7E6,1.7E6,1.7E6 # calibration constants (will be determined<br />
# automatically, if commented out)<br />
dref &nbsp;:= 4.2 # average upper distance limit for<br />
# automatic calibration<br />
peakcheck peaks=$peaks prot=$prot<br />
calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref<br />
peaks calibrate "**" simple<br />
write upl short.upl<br />
</pre> <br />
<br> For the <tt>calibration</tt> parameter you can provide the list of calibration constants you have derived for the "backbone" class with <tt>caliba</tt>, when you calibrated the initial peak lists for use with FOUND/HABAS. Do not comment or delete this line, leave it blank if you want automatic calibration. Automatic calibration uses the <tt>dref</tt> parameter as the presumed average distance for all peaks in a peaklist (not just for backbone, like <tt>caliba</tt>).<br> <br />
<br />
<br> <br />
<br />
=== '''CALC.cya''' ===<br />
<pre>peaks &nbsp;:= n,ali,aro # names of NOESY peak lists<br />
prot &nbsp;:= $name # names of chemical shift lists<br />
constraints&nbsp;:= gridsearch.aco,short.upl,stereofound.cya # additional (non-NOE) constraints<br />
tolerance &nbsp;:= 0.05,0.02,0.4 # chemical shift tolerances<br />
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)<br />
#upl_values &nbsp;:= 2.4,6.0 # calibration cutoffs<br />
calibration&nbsp;:= 1.7E6,1.7E6,1.7E6 # NOE calibration parameters<br />
structures &nbsp;:= 100,20 # number of initial, final structures<br />
steps &nbsp;:= 10000 # number of torsion angle dynamics steps<br />
rmsdrange &nbsp;:= 10..100 # residue range for RMSD calculation<br />
randomseed &nbsp;:= 434726 # random number generator seed<br />
dref &nbsp;:= 4.0 # average distance for calibration, default 4.0<br />
keep &nbsp;:= # set to KEEP to retain existing assignments<br />
<br />
subroutine KEEP<br />
peaks select "*,* number=20000..37999"<br />
end<br />
<br />
#protocol&nbsp;:= noeassign.out # output logging on<br />
noeassign peaks=$peaks prot=$prot calibration=$calibration keep=$keep autoaco<br />
#protocol&nbsp;:=<br />
</pre> <br />
<br> Parameter <tt>constraints</tt> can be a comma-separated list of all kinds of external constraints, which can be read by <tt>read data</tt> command in CYANA. You can have UPLs, ACOs and even .cya scripts, for example, defining stereospecific assignments of methyl groups. Do not comment this line, leave it blank if you are not providing external constraints. <br />
<br />
If you a providing stereospecific assignments, do not use <tt>atom swap</tt> in the <tt>stereofound.cya</tt> script. Use atom list and <tt>short.upl</tt> with all required labels swapped, then the <tt>stereo.cya</tt> should only contain <tt>atom stereo</tt> declarations. <br />
<br />
For the <tt>tolerance</tt> parameter pay attention to the unintuitive dimension order. The recommended tolerances are: 0.03 ppm or less for 1H (0.02 ppm or less for 2D homonuclear peaklists) and 0.6 ppm or less for 15N and 13C. <br />
<br />
Lower and upper limit cutoffs can be changed by applying <tt>upl_values</tt>. The default values are 2.4 and 5.5 A, respectively. <br />
<br />
For the <tt>calibration</tt> parameter you can provide the list of calibration constants you have derived for the "backbone" class with <tt>caliba</tt>, when you calibrated the initial peak lists for use with FOUND/HABAS. Do not comment or delete this line, leave it blank if you want automatic calibration. Automatic calibration uses the <tt>dref</tt> parameter as the presumed average distance for all peaks in a peaklist (not just for backbone, like <tt>caliba</tt>). Having initial calibration too tight is less of an issue with <tt>noeassign</tt>, because by default it "elastically" relaxes constrains, which are consistently violated. <br />
<br />
Use the <tt>protocol</tt> keywords to enable output logging when running CYANA on a workstation. They may not be necessary on a cluster, because the queue system generates its own log. <br />
<br />
Use the subroutine <tt>KEEP</tt> to keep assignment for peaks that you are confident, which is helpful if you peak list contains simulated peaks for short range NOEs. <br />
<br />
=== '''cyana.pbs - PBS queue submission script''' ===<br />
<pre>#!/bin/csh<br />
#!/bin/csh<br />
#PBS -m abe<br />
#PBS -M yourname@domain<br />
#PBS -q short_c<br />
#PBS -l nodes=5:ppn=2<br />
#PBS -l walltime=02:00:00<br />
#PBS -o cyana.out<br />
#PBS -j oe<br />
#PBS -N cyana<br />
#<br />
cd $PBS_O_WORKDIR<br />
echo "working directory = "$PBS_O_WORKDIR<br />
set NN = `cat $PBS_NODEFILE | wc -l`<br />
echo "NN = "$NN<br />
module load mpich/intel-9/ch_p4/current<br />
module load cyana/2.1-p4<br />
limit stacksize unlimited<br />
limit coredumpsize 0<br />
source $MODULESHOME/init/tcsh<br />
cat $PBS_NODEFILE | awk '{printf "%s.ccr.buffalo.edu\n",$1}' &gt; tmp.$$<br />
cyana -c '/util/mpich/1.2.7p1/intel-9/ch_p4/bin/mpiexec ' ./CALC<br />
#<br />
echo "ALL Done!"<br />
</pre> <br />
<br> The <tt>#PBS</tt> lines pass option to the PBS queue system. See [http://www.ccr.buffalo.edu/hotpages/content/pbsEXia32.htm this page] for details <br />
<br />
The following options are important: <br />
<br />
*<br />
<br />
<tt>#PBS -m abe</tt> tell PBS queue system to send e-mail alerts when calculation starts (<tt>b), aborts (a</tt>) or terminates successfully (e). <br />
<br />
*Enter you e-mail address in <tt>#PBS -M myname@mydomain</tt>. Without this line e-mail alerts will go into local mailbox. <br />
*The line <tt>#PBS -l nodes=5:ppn=2</tt> means that we are using five dual-processor nodes and get 10-fold parallelization during simulated annealing. It doesn't make much sense to request more than 5 nodes: first, the relative gain in speed drops since NOE assignment step cannot be parallelized; second, the queue wait time may be longer when more nodes are requested. <br />
*<tt>#PBS -q short_c</tt> submits the job to the <tt>short_c</tt> queue. This queue is dedicated to short jobs and has higher priority. Members of Szyperski's lab have 10 nodes reserved for this queue every weekday 9 a.m. - 4 p.m. <br />
*<tt>#PBS -l walltime=02:00:00</tt> defines maximum allocated job execution time. The limit for the <tt>shorts_c</tt> queue is 2 hours, but even the most demanding CYANA job finish in less than one hour.<br />
<br />
<br><br><br />
<br />
*[[Media:CALC_noeassign.cya|CALC.cya]]: CYANA 2.1 automated structure calculation script<br />
<br />
*[[Media:Cyana.pbs|cyana.pbs]]: PBS queue submission script for CYANA 2.1 on U2 cluster<br />
<br />
*[[Media:Make_short.cya|make_short.cya]]: CYANA script to run manual calculation with local constraints</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=Manual_Structure_Calculation_Using_CYANA&diff=3591
Manual Structure Calculation Using CYANA
2010-01-06T21:54:13Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
Here we describe structure calcuation with manual NOE asignments using CYANA 2.1.&nbsp; A tutorial is also avaliable [http://www.cyana.org/wiki/index.php/Structure_calculation_using_manually_assigned_NOESY_peak_lists on-line] for manual structure calculation using CYANA 3.0. <br />
<br />
== '''Protocol''' ==<br />
<br />
Manual structure calculation is performed similar to the [[NoeassignCYANARun|automated calculation]]. <br />
<br />
#Create a new working subdirectory (<tt>structure/cyana21/calc3</tt>) <br />
#Copy init.cya, XXXX.seq, XXXX.prot into this directiory. <br />
#Export the latest peaklists, like n-cons, ali-cons and aro-cons. <br />
#Copy the latest <tt>stereofound.cya</tt> from the latest GridSearch run. <br />
#(Optional) Copy the <tt>talos.aco</tt> file <br />
#Download the [[Media:CALC_manual.cya|CALC.cya]] file (see below). Modify peaklist names, calibration parameters and tolerances. <br />
#Run calculation with CYANA 2.1 on a workstation or U2 cluster<br />
<br />
<br> <br />
<br />
== '''CALC.cya''' ==<br />
<pre>peaks &nbsp;:= n_cons,ali_cons,aro_cons # names of peak lists<br />
prot &nbsp;:= $name # names of proton lists<br />
tolerance &nbsp;:= 0.05,0.02,0.4 # chemical shift tolerances<br />
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)<br />
if (master) then<br />
# ---- check consistency of peak and chemical shift lists----<br />
peakcheck peaks=$peaks prot=$prot<br />
# ---- calibration ----<br />
read prot $prot<br />
read peaks n_cons<br />
peaks set volume=abs(volume)<br />
caliba bb=2.0E+06 sc=2.0E+05 methyl=4.0E+04<br />
write upl n<br />
read peaks ali_cons<br />
peaks set volume=abs(volume)<br />
caliba bb=2.5E+06 sc=4.3E+05 methyl=2.0E+05<br />
write upl ali<br />
read peaks aro_cons<br />
peaks set volume=abs(volume)<br />
caliba bb=1.7E+06 sc=2.4E+05 methyl=6.0E+04<br />
<br />
write upl $name-in.upl<br />
stereofound<br />
distance modify<br />
write upl $name.upl<br />
end if<br />
synchronize<br />
<br />
# ---- structure calculation ----<br />
expand=0<br />
read seq $name.seq # re-read sequence to initialize<br />
read upl $name.upl # read upper distance limits<br />
#read aco talos.aco # read angle constraints<br />
seed=5671 # random number generator seed<br />
calc_all structures=100 command=anneal steps=10000 # calculate 100 conformers<br />
flip # align side chains for lower RMSD<br />
overview $name.ovw structures=20 pdb # write overview file and coordinates<br />
#overview $name.ovw structures=20 significant=1 pdb<br />
</pre> <br />
<br> Here <tt>expand=0</tt> is needed to correctly interpret UPLs created with CALIBA. <br />
<br />
Macro <tt>flip.cya</tt> can be used to flip the side chains Glu, Asp, Phe and Tyr to reduce RMSD in the final calculation. <br />
<br />
Overview by default will report only violation in at least <tt>structures/3</tt> conformers, and in this particular case these are six. To have all violations reported add <tt>significant=1</tt>. <br />
<br />
Macro <tt>peakcheck</tt> verifies AtomList and peaklists for consistency and is very useful when manually assigned peaks are used. <br />
<br />
It is important to set peak volumes to their absolute values before calling caliba for calibration.</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=NOE_Calibration_Using_CYANA&diff=3590
NOE Calibration Using CYANA
2010-01-06T21:53:43Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
NOE calibration converts NOESY cross-peak intensities into upper distance limits. A certain functional relationship is assumed between peak intensities and UPLs for a given group of peaks. The most common is NOE peak volume proportional to interproton distance to the minus six: <br> <br />
<pre> V = A/d^6</pre> <br />
where <tt>V</tt> is the peak volume (or intensity), <tt>d</tt> is the upper distance limit, and <tt>A</tt> is the calibration constant. <br />
<br />
<br> <br />
<br />
== '''NOE Calibration Using CYANA 2.1''' ==<br />
<br />
This section describes how to determine NOE calibration constants using CYANA 2.1. <br />
<br />
Before running the calibration, the N chemical shifts in the peak list must be updated, and the peak lists separated into a list of N noes and C noes, using the UBNMR macro, '''macro1'''. <br />
<br />
=== '''Command "calibrate"''' ===<br />
<br />
A CYANA command <tt>calibrate</tt> is the basic calibration tool, unchanged since DYANA. <br />
<br />
<br> <br />
<pre> Parameters: f(d) (required)<br />
dmin [d2 ...] dmax (default: 2.4 5.5)<br />
weight=w (default: 1.0)<br />
plot=file log minimal (default: none)<br />
</pre> <br />
<tt>calibrate</tt> accepts a calibration function <tt>f(d)</tt> as the required argument. <br />
<br />
This command uses "center" averaging. Explicit pseudoatom corrections are added to UPLs: <br />
<br />
*Multiplicity correction is applied by dividing the peak volume by the numbers of 1H spins in pseudoatoms assigned to the peak. For instance, the volume of a cross peak between a Leu QQD pseudo atom and a Tyr QD pseudo atom is divided by a factor of 6 x 2 = 12 prior to applying the calibration function. The resulting UPL is the subject to the upper and lower cutoffs. <br />
*Distance correction is applied by adding a distance between the pseudoatom and its constituent spins. It is applied after the application of upper and lower cutoffs. For example, for a Tyr QD pseudoatom this correction is equal to half the distance between the HD1 and HD2 spins. Distance correction can be manually performed with the <tt>distance correct</tt> command.<br />
<br />
Type <tt>help calibrate</tt> at CYANA prompt for more information or see the DYANA documentation. <br />
<br />
<br> <br />
<br />
=== '''Macro "caliba"''' ===<br />
<br />
CYANA macro <tt>caliba.cya</tt> is functionally the same as <tt>caliba.dya</tt> of DYANA. It is a more sophisticated calibration tool aimed at getting more nuanced calibration than a uniform <code>1/d</code><sup><code>6</code></sup> dependence. CALIBA is recommended for manual structure calculation with CYANA 2.1. The <tt>caliba</tt> macro in fact uses <tt>calibrate</tt>, therefore, it also uses "center" averaging. <br />
<br />
<br> <br />
<pre>Caliba parameters:<br />
dmin=dmin (default: 2.4)<br />
dmax=dmax (default: 5.5)<br />
vmin=Vmin (default: 0.0)<br />
bb=A (default: calculated automatically)<br />
sc=B (default: A/dmin2)<br />
methyl=C (default: B/3)<br />
weight=w (default: 1.0)<br />
avedis=d (default: 3.4)<br />
plot=file (optional)<br />
</pre> <br />
This macro divides each peaklist into three classes of peaks with different calibration functions and constants: <br />
<br />
<br> <br />
<br />
{| rules="rows" cellspacing="0" cellpadding="0" border="1" class="twikiTable" id="table1" style="width: 638px; height: 76px;"<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
! valign="top" bgcolor="#687684" class="twikiTableCol0 twikiFirstCol" | <font color="#ffffff">class</font> <br />
! valign="top" bgcolor="#687684" class="twikiTableCol1" | <font color="#ffffff">peaks/constraints</font> <br />
! valign="top" bgcolor="#687684" class="twikiTableCol2 twikiLastCol" | <font color="#ffffff">function</font><br />
|- class="twikiTableEven twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol" | <br />
backbone&nbsp; <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; all HN/HA— HN/HA, and HN(i)/HA(i) — HB(j) with abs(i - j) &lt; 5&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2 twikiLastCol" | <br />
<code></code><code>V = A/d</code><sup><code>6</code></sup> <br />
<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted1 twikiTableRowdataBg1"<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol0 twikiFirstCol" | <br />
sidechain <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; neither "backbone" nor "methyl" <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol2 twikiLastCol" | <br />
<code>V = B/d<sup>4</sup></code><code></code> <br />
<br />
|- class="twikiTableEven twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol twikiLast" | <br />
methyl <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1 twikiLast" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; all involving methyl groups <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2 twikiLastCol twikiLast" | <br />
<code>V = C/d<sup>4</sup></code><code></code> <br />
<br />
|}<br />
<br />
<br> <br />
<br />
By default, constant <tt>A</tt> is calculated from <tt>avedis</tt> - the assumed average distance between backbone atoms. It is also possible to specify the backbone constant <tt>A</tt> explicitly. Constant <tt>B</tt> is then calculated from <tt>A</tt>, and <tt>C</tt> from <tt>B</tt> <br />
<br />
Parameters <tt>dmin</tt> and <tt>dmax</tt> are the lower and upper cutoff limits for UPLs, respectively. <br />
<br />
Parameter <tt>vmin</tt> is useful to filter out peaks with intensities below a certain threshold. Without it even incorrectly picked peaks with intensities below S/N level will produce UPLs set at the upper cutoff. <br />
<br />
<tt>plot</tt> is an optional parameter, used to create logarithmic plots of volumes versus corresponding minimal distances in the selected structures together with the calibration functions. It is useful for fine-tuning the calibration during structure refinement. Of course, a structure must be then loaded into CYANA before running <tt>caliba</tt>. <br />
<br />
<br> '''VERY IMPORTANT!!!'''. The default behavior of structure calculation (<tt>anneal</tt>, <tt>calc_all</tt>, <tt>noeassign</tt>) in CYANA 2.x is to use "sum of r<sup>-6</sup>" averaging for UPLs. Setting <tt>expand=1</tt> declares "sum of r<sup>-6</sup>" averaging, and <tt>expand=0</tt> declares "center" averaging. If your UPLs reflect "center" averaging (that is, they were created with <tt>caliba</tt> or <tt>calibrate</tt>), then <br />
<br />
*They can be used with DYANA, CYANA 1.x and AutoStructure. <br />
*They can be used with legacy macros in CYANA 2.1, such as <tt>habas</tt>. <br />
*They can be used for manual structure calculation in CYANA 2.1, if <tt>expand=0</tt> is set before calling <code>anneal</code>. <br />
*'''Do not''' use them for automated structure calculation in CYANA 2.1 with <tt>noeassign</tt>. The <tt>noeassign</tt> macro employs <tt>peaks calibrate</tt> (see below) and sets <tt>expand=1</tt>. In general, '''do not''' mix them with UPLs calibrated with <tt>peaks calibrate</tt>.<br />
<br />
Type <tt>help caliba</tt> at CYANA prompt or see the DYANA manual for more information. You can also look at the script itself in the <tt>macro</tt> subdirectory of a CYANA installation. <br />
<br />
<br> <br />
<br />
=== '''Command "peaks calibrate"''' ===<br />
<br />
Command <tt>peaks calibrate</tt> is a new feature of CYANA 2.x, which produced a "sum of r<sup>-6</sup>"-averaged calibration. The documentation for it is scarce. For example, the manual structure calculation script <tt>~/demo/manual/CALC.cya</tt> contains these lines: <br />
<br />
<br> <br />
<pre>peaks &nbsp;:= c13,n15,aro # names of peak lists<br />
prot &nbsp;:= demo # names of proton lists<br />
tolerance &nbsp;:= 0.040,0.030,0.45 # chemical shift tolerances<br />
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)<br />
calibration:= 6.7E5,8.2E5,8.0E4 # calibration constants (will be determined<br />
# automatically, if commented out)<br />
dref &nbsp;:= 4.2 # average upper distance limit for<br />
# automatic calibration<br />
<br />
calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref<br />
peaks calibrate "**" simple<br />
write upl $name-in.upl<br />
</pre> <br />
Line <tt>calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref</tt> calls the macro <tt>calibration.cya</tt>, which iteratively sets calibration constants for each peaklist. If the <tt>calibration</tt> list variable is defined, its values are taken as calibration constants. If it is left blank, calibration constants are calibrated based on assumed average distance <tt>dref</tt> between '''all''' atoms of the molecule. Note that <tt>dref</tt> here is inherently different from <tt>avedis</tt> of <tt>caliba</tt> - the latter refers only to distances between <tt>backbone</tt> protons. <br />
<br />
Line <tt>peaks calibrate "**" simple</tt> generates UPLs assuming simple <tt>C/d^6</tt> functions with calibration constants <tt>C</tt> from the previous step. These calibration constants are thus closely related the "backbone" class constants <tt>A</tt> of <tt>caliba</tt>. <br />
<br />
To modify upper and lower cutoffs for NOE calibration with <tt>peaks calibrate</tt> add a line <tt>set upl_values:=2.4,6.0</tt>. The defaults are 2.4 and 5.5. <br />
<br />
The main difference from <tt>calibrate</tt> and <tt>caliba</tt> is that pseudoatom correction are '''not applied''' to resulting UPLs. <br />
<br />
<br> <br />
<br />
'''VERY IMPORTANT!!!'''. The default behavior of structure calculation (<tt>anneal</tt>, <tt>calc_all</tt>, <tt>noeassign</tt>) in CYANA 2.x is to use "sum of r<sup>-6</sup>" averaging for UPLs. Setting <tt>expand=1</tt> declares "sum of r<sup>-6</sup>" averaging, and <tt>expand=0</tt> declares "center" averaging. If you UPLs use "sum of r<sup>-6</sup>" averaging (if created with <tt>peaks calibrate</tt>), then <br />
<br />
*'''Do not''' use them with DYANA, CYANA 1.x and AutoStructure. <br />
*'''Do not''' use them with legacy macros in CYANA 2.1, such as <tt>habas</tt>. <br />
*They can be used for manual structure calculation in CYANA 2.1, if <tt>expand=1</tt> is set before calling <tt>anneal</tt>. <br />
*They can be used for automated structure calculation in CYANA 2.1 with <tt>noeassign</tt>. The <tt>noeassign</tt> macro employs <tt>peaks calibrate</tt> (see below) and sets <tt>expand=1</tt>.<br />
<br />
<br> <br />
<br />
The aim of this new calibration method is likely to facilitate automated NOE assignment and structure calculation with <tt>noeassign</tt>. Unlike <tt>caliba</tt> and <tt>calibrate</tt>, <tt>peaks calibrate</tt> effectively generates UPLs even for unassigned peaks assuming NOEs between single protons. If such a peak gets assigned to a pseudoatom, the required correction is applied implicitly during the calculation. <br />
<br />
<br> <br />
<br />
=== Initial NOE&nbsp;Calibration ===<br />
<br />
After simulating and filtering one typically has three initial NOESY peaklists, for example, <tt>n.peaks</tt>, <tt>ali.peaks</tt>, and <tt>aro.peaks</tt>. The assigned peaks are intra-residue and short-range within secondary structure elements. Initial calibration is needed to verify these assignments for consistency with molecular geometry and, if possible, to derive stereospecific assignments with HABAS in CYANA. If your SequenceList and AtomList were generated with XEASY, you will need to convert them to the format of CYANA 2.1 (see the <tt>~/demo/details/MigrateFromDyanaCyana1.cya</tt> example script). SequenceList and AtomList from CARA are already compatible. <br />
<br />
Amide, aliphatic and aromatic protons tend to have different relaxation properties in a simultaneous 3D <sup>15</sup>N-, <sup>13</sup>C-resolved NOESY.&nbsp; NOESY spectra acquired separately may have varying acquisition times. Therefore, it is recommended to run <tt>caliba</tt> separately for each peaklist. <br />
<br />
Here is a sample [[Media:Run_caliba.cya|run_caliba.cya]] script for running <tt>caliba</tt> on all three peaklists: <br />
<pre>protocol:=caliba.log<br />
read prot $name<br />
<br />
read peaks n<br />
peaks select "**"<br />
peaks set volume=abs(volume)<br />
caliba<br />
<br />
read peaks ali<br />
peaks select "**"<br />
peaks set volume=abs(volume)<br />
caliba<br />
<br />
read peaks aro<br />
peaks select "**"<br />
peaks set volume=abs(volume)<br />
caliba bb=1.0E+06<br />
<br />
write upl short.upl<br />
protocol:=</pre> <br />
Note that you need to convert volumes to absolute values before calling <tt>caliba</tt>, otherwise negative intensities will be misinterpreted. <br />
<br />
Macro <tt>caliba</tt> should work fine for <tt>n.peaks</tt> and <tt>ali.peaks</tt> with default parameters. Aromatic peaklist <tt>aro.peaks</tt> requires special treatment because it does not have any peaks of the backbone class. '''For aromatic peaklists <tt>caliba</tt> should be run either with a single <tt>bb</tt> parameter or with both <tt>bb</tt> and <tt>sc</tt> parameters. Calling it without <tt>bb</tt> parameter will cause an error.''' <br />
<br />
The <tt>sc</tt> and <tt>bb</tt> parameters for <tt>aro.peaks</tt> can be estimated from the aliphatic calibration. Typically the aromatic part of the simultaneous NOESY spectrum is less sensitive than the aromatic, therefore the values should be somewhat lower. <br />
<br />
<br> <br />
<br />
=== Verifying Initial NOE&nbsp;Calibration ===<br />
<br />
Inspect resulting UPL files to make sure the the calibration is correct. You should avoid making UPLs too tight or too loose. <br />
<br />
Known short-range distances in the secondary structure elements constitute good reference points. Consult the table below (adapted in short form from Ref. 1). <br />
<br />
<br> <br />
<br />
{| rules="rows" cellspacing="0" cellpadding="0" border="1" class="twikiTable" id="table2" style="width: 556px; height: 151px;"<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
! valign="top" bgcolor="#687684" class="twikiTableCol0 twikiFirstCol" | <br />
<font color="#ffffff">distance</font><font color="#ffffff"></font> <br />
<br />
! valign="top" bgcolor="#687684" class="twikiTableCol1" | <br />
<font color="#ffffff">α-helix</font><font color="#ffffff"></font><br> <br />
<br />
! valign="top" bgcolor="#687684" class="twikiTableCol2" | <br />
<font color="#ffffff">3_10-helix</font><font color="#ffffff"></font><br> <br />
<br />
! valign="top" bgcolor="#687684" class="twikiTableCol3" | <br />
<font color="#ffffff">β-sheet (A)</font><font color="#ffffff"></font><br> <br />
<br />
! valign="top" bgcolor="#687684" class="twikiTableCol4 twikiLastCol" | <br />
[http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/NOECalibrationCYANA?sortcol=4;table=2;up=0#sorted_table <font color="#ffffff">β-sheet (P)</font>]<font color="#ffffff"></font><br> <br />
<br />
|- class="twikiTableEven twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol" | <br />
d_αN<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 3.5<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3.4<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol3" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.2<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol4 twikiLastCol" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.2<br> <br />
<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted1 twikiTableRowdataBg1"<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol0 twikiFirstCol" | <br />
d_αN(i,i+2)<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 4.4<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol2" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3.8<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol3" | &nbsp; <br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol4 twikiLastCol" | &nbsp;<br />
|- class="twikiTableEven twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol" | <br />
d_αN(i,i+3)<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 3.4<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3.3<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol3" | &nbsp; <br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol4 twikiLastCol" | &nbsp;<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted1 twikiTableRowdataBg1"<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol0 twikiFirstCol" | <br />
d_αN(i,i+4)<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol1" | <br />
&nbsp;&nbsp; &nbsp; 4.2<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol2" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3.3<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol3" | &nbsp; <br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol4 twikiLastCol" | &nbsp;<br />
|- class="twikiTableEven twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol" | <br />
d_NN<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 2.8<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.6<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol3" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4.3<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol4 twikiLastCol" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4.2<br> <br />
<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted1 twikiTableRowdataBg1"<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol0 twikiFirstCol twikiLast" | <br />
d_NN(i,i+2)<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol1 twikiLast" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 4.2<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol2 twikiLast" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4.1<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol3 twikiLast" | &nbsp; <br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol4 twikiLastCol twikiLast" | &nbsp;<br />
|}<br />
<br />
For calibration of aromatic peaklists the characteristic distances are: <br />
<br />
<br> <br />
<br />
{| rules="rows" cellspacing="0" cellpadding="0" border="1" class="twikiTable" id="table3" style="width: 285px; height: 106px;"<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol" | <br> <br />
! valign="top" bgcolor="#687684" class="twikiTableCol1" | <br />
<font color="#ffffff">distance</font><font color="#ffffff"></font> <br />
<br />
! valign="top" bgcolor="#687684" class="twikiTableCol2 twikiLastCol" | <br />
<font color="#ffffff">center averaging</font><font color="#ffffff"></font> <br />
<br />
|- class="twikiTableEven twikiTableRowdataBgSorted1 twikiTableRowdataBg1"<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol0 twikiFirstCol" | <br />
d_δβ <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.33 - 3.77 <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol2 twikiLastCol" | <br />
5.00 - 6.75 <br />
<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
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d_δε <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1 twikiLast" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.48 <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2 twikiLastCol twikiLast" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 7.4 <br />
<br />
|}<br />
<br />
Note that aromatic QD and QE are pseudoatoms and the constraints in "center" averaging mode are much larger than the distances between individual atoms. <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
== '''References''' ==<br />
<br />
1.&nbsp;&nbsp; Wuthrich, K. (1986)&nbsp; "NMR of Proteins and Nucleic Acids", page 127. <br />
<br />
<br> <br />
<br />
<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=NOE_Calibration_Using_CYANA&diff=3589
NOE Calibration Using CYANA
2010-01-06T21:53:03Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
NOE calibration converts NOESY cross-peak intensities into upper distance limits. A certain functional relationship is assumed between peak intensities and UPLs for a given group of peaks. The most common is NOE peak volume proportional to interproton distance to the minus six: <br> <br />
<pre> V = A/d^6</pre> <br />
where <tt>V</tt> is the peak volume (or intensity), <tt>d</tt> is the upper distance limit, and <tt>A</tt> is the calibration constant. <br />
<br />
<br> <br />
<br />
== '''NOE Calibration Using CYANA 2.1''' ==<br />
<br />
This section describes how to determine NOE calibration constants using CYANA 2.1. <br />
<br />
Before running the calibration, the N chemical shifts in the peak list must be updated, and the peak lists separated into a list of N noes and C noes, using the UBNMR macro, '''macro1'''. <br />
<br />
=== '''Command "calibrate"''' ===<br />
<br />
A CYANA command <tt>calibrate</tt> is the basic calibration tool, unchanged since DYANA. <br />
<br />
<br> <br />
<pre> Parameters: f(d) (required)<br />
dmin [d2 ...] dmax (default: 2.4 5.5)<br />
weight=w (default: 1.0)<br />
plot=file log minimal (default: none)<br />
</pre> <br />
<tt>calibrate</tt> accepts a calibration function <tt>f(d)</tt> as the required argument. <br />
<br />
This command uses "center" averaging. Explicit pseudoatom corrections are added to UPLs: <br />
<br />
*Multiplicity correction is applied by dividing the peak volume by the numbers of 1H spins in pseudoatoms assigned to the peak. For instance, the volume of a cross peak between a Leu QQD pseudo atom and a Tyr QD pseudo atom is divided by a factor of 6 x 2 = 12 prior to applying the calibration function. The resulting UPL is the subject to the upper and lower cutoffs. <br />
*Distance correction is applied by adding a distance between the pseudoatom and its constituent spins. It is applied after the application of upper and lower cutoffs. For example, for a Tyr QD pseudoatom this correction is equal to half the distance between the HD1 and HD2 spins. Distance correction can be manually performed with the <tt>distance correct</tt> command.<br />
<br />
Type <tt>help calibrate</tt> at CYANA prompt for more information or see the DYANA documentation. <br />
<br />
<br> <br />
<br />
=== '''Macro "caliba"''' ===<br />
<br />
CYANA macro <tt>caliba.cya</tt> is functionally the same as <tt>caliba.dya</tt> of DYANA. It is a more sophisticated calibration tool aimed at getting more nuanced calibration than a uniform <code>1/d</code><sup><code>6</code></sup> dependence. CALIBA is recommended for manual structure calculation with CYANA 2.1. The <tt>caliba</tt> macro in fact uses <tt>calibrate</tt>, therefore, it also uses "center" averaging. <br />
<br />
<br> <br />
<pre>Caliba parameters:<br />
dmin=dmin (default: 2.4)<br />
dmax=dmax (default: 5.5)<br />
vmin=Vmin (default: 0.0)<br />
bb=A (default: calculated automatically)<br />
sc=B (default: A/dmin2)<br />
methyl=C (default: B/3)<br />
weight=w (default: 1.0)<br />
avedis=d (default: 3.4)<br />
plot=file (optional)<br />
</pre> <br />
This macro divides each peaklist into three classes of peaks with different calibration functions and constants: <br />
<br />
<br> <br />
<br />
{| rules="rows" cellspacing="0" cellpadding="0" border="1" style="width: 638px; height: 76px;" id="table1" class="twikiTable"<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
! valign="top" bgcolor="#687684" class="twikiTableCol0 twikiFirstCol" | <font color="#ffffff">class</font> <br />
! valign="top" bgcolor="#687684" class="twikiTableCol1" | <font color="#ffffff">peaks/constraints</font> <br />
! valign="top" bgcolor="#687684" class="twikiTableCol2 twikiLastCol" | <font color="#ffffff">function</font><br />
|- class="twikiTableEven twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol" | <br />
backbone&nbsp; <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; all HN/HA— HN/HA, and HN(i)/HA(i) — HB(j) with abs(i - j) &lt; 5&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2 twikiLastCol" | <br />
<code></code><code>V = A/d</code><sup><code>6</code></sup> <br />
<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted1 twikiTableRowdataBg1"<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol0 twikiFirstCol" | <br />
sidechain <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; neither "backbone" nor "methyl" <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol2 twikiLastCol" | <br />
<code>V = B/d<sup>4</sup></code><code></code> <br />
<br />
|- class="twikiTableEven twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol twikiLast" | <br />
methyl <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1 twikiLast" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; all involving methyl groups <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2 twikiLastCol twikiLast" | <br />
<code>V = C/d<sup>4</sup></code><code></code> <br />
<br />
|}<br />
<br />
<br> <br />
<br />
By default, constant <tt>A</tt> is calculated from <tt>avedis</tt> - the assumed average distance between backbone atoms. It is also possible to specify the backbone constant <tt>A</tt> explicitly. Constant <tt>B</tt> is then calculated from <tt>A</tt>, and <tt>C</tt> from <tt>B</tt> <br />
<br />
Parameters <tt>dmin</tt> and <tt>dmax</tt> are the lower and upper cutoff limits for UPLs, respectively. <br />
<br />
Parameter <tt>vmin</tt> is useful to filter out peaks with intensities below a certain threshold. Without it even incorrectly picked peaks with intensities below S/N level will produce UPLs set at the upper cutoff. <br />
<br />
<tt>plot</tt> is an optional parameter, used to create logarithmic plots of volumes versus corresponding minimal distances in the selected structures together with the calibration functions. It is useful for fine-tuning the calibration during structure refinement. Of course, a structure must be then loaded into CYANA before running <tt>caliba</tt>. <br />
<br />
<br> '''VERY IMPORTANT!!!'''. The default behavior of structure calculation (<tt>anneal</tt>, <tt>calc_all</tt>, <tt>noeassign</tt>) in CYANA 2.x is to use "sum of r<sup>-6</sup>" averaging for UPLs. Setting <tt>expand=1</tt> declares "sum of r<sup>-6</sup>" averaging, and <tt>expand=0</tt> declares "center" averaging. If your UPLs reflect "center" averaging (that is, they were created with <tt>caliba</tt> or <tt>calibrate</tt>), then <br />
<br />
*They can be used with DYANA, CYANA 1.x and AutoStructure. <br />
*They can be used with legacy macros in CYANA 2.1, such as <tt>habas</tt>. <br />
*They can be used for manual structure calculation in CYANA 2.1, if <tt>expand=0</tt> is set before calling <code>anneal</code>. <br />
*'''Do not''' use them for automated structure calculation in CYANA 2.1 with <tt>noeassign</tt>. The <tt>noeassign</tt> macro employs <tt>peaks calibrate</tt> (see below) and sets <tt>expand=1</tt>. In general, '''do not''' mix them with UPLs calibrated with <tt>peaks calibrate</tt>.<br />
<br />
Type <tt>help caliba</tt> at CYANA prompt or see the DYANA manual for more information. You can also look at the script itself in the <tt>macro</tt> subdirectory of a CYANA installation. <br />
<br />
<br> <br />
<br />
=== '''Command "peaks calibrate"''' ===<br />
<br />
Command <tt>peaks calibrate</tt> is a new feature of CYANA 2.x, which produced a "sum of r<sup>-6</sup>"-averaged calibration. The documentation for it is scarce. For example, the manual structure calculation script <tt>~/demo/manual/CALC.cya</tt> contains these lines: <br />
<br />
<br> <br />
<pre>peaks &nbsp;:= c13,n15,aro # names of peak lists<br />
prot &nbsp;:= demo # names of proton lists<br />
tolerance &nbsp;:= 0.040,0.030,0.45 # chemical shift tolerances<br />
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)<br />
calibration:= 6.7E5,8.2E5,8.0E4 # calibration constants (will be determined<br />
# automatically, if commented out)<br />
dref &nbsp;:= 4.2 # average upper distance limit for<br />
# automatic calibration<br />
<br />
calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref<br />
peaks calibrate "**" simple<br />
write upl $name-in.upl<br />
</pre> <br />
Line <tt>calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref</tt> calls the macro <tt>calibration.cya</tt>, which iteratively sets calibration constants for each peaklist. If the <tt>calibration</tt> list variable is defined, its values are taken as calibration constants. If it is left blank, calibration constants are calibrated based on assumed average distance <tt>dref</tt> between '''all''' atoms of the molecule. Note that <tt>dref</tt> here is inherently different from <tt>avedis</tt> of <tt>caliba</tt> - the latter refers only to distances between <tt>backbone</tt> protons. <br />
<br />
Line <tt>peaks calibrate "**" simple</tt> generates UPLs assuming simple <tt>C/d^6</tt> functions with calibration constants <tt>C</tt> from the previous step. These calibration constants are thus closely related the "backbone" class constants <tt>A</tt> of <tt>caliba</tt>. <br />
<br />
To modify upper and lower cutoffs for NOE calibration with <tt>peaks calibrate</tt> add a line <tt>set upl_values:=2.4,6.0</tt>. The defaults are 2.4 and 5.5. <br />
<br />
The main difference from <tt>calibrate</tt> and <tt>caliba</tt> is that pseudoatom correction are '''not applied''' to resulting UPLs. <br />
<br />
<br> <br />
<br />
'''VERY IMPORTANT!!!'''. The default behavior of structure calculation (<tt>anneal</tt>, <tt>calc_all</tt>, <tt>noeassign</tt>) in CYANA 2.x is to use "sum of r<sup>-6</sup>" averaging for UPLs. Setting <tt>expand=1</tt> declares "sum of r<sup>-6</sup>" averaging, and <tt>expand=0</tt> declares "center" averaging. If you UPLs use "sum of r<sup>-6</sup>" averaging (if created with <tt>peaks calibrate</tt>), then <br />
<br />
*'''Do not''' use them with DYANA, CYANA 1.x and AutoStructure. <br />
*'''Do not''' use them with legacy macros in CYANA 2.1, such as <tt>habas</tt>. <br />
*They can be used for manual structure calculation in CYANA 2.1, if <tt>expand=1</tt> is set before calling <tt>anneal</tt>. <br />
*They can be used for automated structure calculation in CYANA 2.1 with <tt>noeassign</tt>. The <tt>noeassign</tt> macro employs <tt>peaks calibrate</tt> (see below) and sets <tt>expand=1</tt>.<br />
<br />
<br> <br />
<br />
The aim of this new calibration method is likely to facilitate automated NOE assignment and structure calculation with <tt>noeassign</tt>. Unlike <tt>caliba</tt> and <tt>calibrate</tt>, <tt>peaks calibrate</tt> effectively generates UPLs even for unassigned peaks assuming NOEs between single protons. If such a peak gets assigned to a pseudoatom, the required correction is applied implicitly during the calculation. <br />
<br />
<br> <br />
<br />
=== Initial NOE&nbsp;Calibration ===<br />
<br />
After simulating and filtering one typically has three initial NOESY peaklists, for example, <tt>n.peaks</tt>, <tt>ali.peaks</tt>, and <tt>aro.peaks</tt>. The assigned peaks are intra-residue and short-range within secondary structure elements. Initial calibration is needed to verify these assignments for consistency with molecular geometry and, if possible, to derive stereospecific assignments with HABAS in CYANA. If your SequenceList and AtomList were generated with XEASY, you will need to convert them to the format of CYANA 2.1 (see the <tt>~/demo/details/MigrateFromDyanaCyana1.cya</tt> example script). SequenceList and AtomList from CARA are already compatible. <br />
<br />
Amide, aliphatic and aromatic protons tend to have different relaxation properties in a simultaneous 3D <sup>15</sup>N-, <sup>13</sup>C-resolved NOESY.&nbsp; NOESY spectra acquired separately may have varying acquisition times. Therefore, it is recommended to run <tt>caliba</tt> separately for each peaklist. <br />
<br />
Here is a sample [[Media:Run_caliba.cya|run_caliba.cya]] script for running <tt>caliba</tt> on all three peaklists: <br />
<pre>protocol:=caliba.log<br />
read prot $name<br />
<br />
read peaks n<br />
peaks select "**"<br />
peaks set volume=abs(volume)<br />
caliba<br />
<br />
read peaks ali<br />
peaks select "**"<br />
peaks set volume=abs(volume)<br />
caliba<br />
<br />
read peaks aro<br />
peaks select "**"<br />
peaks set volume=abs(volume)<br />
caliba bb=1.0E+06<br />
<br />
write upl short.upl<br />
protocol:=</pre> <br />
Note that you need to convert volumes to absolute values before calling <tt>caliba</tt>, otherwise negative intensities will be misinterpreted. <br />
<br />
Macro <tt>caliba</tt> should work fine for <tt>n.peaks</tt> and <tt>ali.peaks</tt> with default parameters. Aromatic peaklist <tt>aro.peaks</tt> requires special treatment because it does not have any peaks of the backbone class. '''For aromatic peaklists <tt>caliba</tt> should be run either with a single <tt>bb</tt> parameter or with both <tt>bb</tt> and <tt>sc</tt> parameters. Calling it without <tt>bb</tt> parameter will cause an error.''' <br />
<br />
The <tt>sc</tt> and <tt>bb</tt> parameters for <tt>aro.peaks</tt> can be estimated from the aliphatic calibration. Typically the aromatic part of the simultaneous NOESY spectrum is less sensitive than the aromatic, therefore the values should be somewhat lower. <br />
<br />
<br> <br />
<br />
=== Verifying Initial NOE&nbsp;Calibration ===<br />
<br />
Inspect resulting UPL files to make sure the the calibration is correct. You should avoid making UPLs too tight or too loose. <br />
<br />
Known short-range distances in the secondary structure elements constitute good reference points. Consult the table below (adapted in short form from Ref. 1). <br />
<br />
<br> <br />
<br />
{| rules="rows" cellspacing="0" cellpadding="0" border="1" style="width: 556px; height: 151px;" id="table2" class="twikiTable"<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
! valign="top" bgcolor="#687684" class="twikiTableCol0 twikiFirstCol" | <br />
<font color="#ffffff">distance</font><font color="#ffffff"></font> <br />
<br />
! valign="top" bgcolor="#687684" class="twikiTableCol1" | <br />
<font color="#ffffff">α-helix</font><font color="#ffffff"></font><br> <br />
<br />
! valign="top" bgcolor="#687684" class="twikiTableCol2" | <br />
<font color="#ffffff">3_10-helix</font><font color="#ffffff"></font><br> <br />
<br />
! valign="top" bgcolor="#687684" class="twikiTableCol3" | <br />
<font color="#ffffff">β-sheet (A)</font><font color="#ffffff"></font><br> <br />
<br />
! valign="top" bgcolor="#687684" class="twikiTableCol4 twikiLastCol" | <br />
[http://www.nsm.buffalo.edu/Chem/HTP_twiki43/bin/view/NESG/NOECalibrationCYANA?sortcol=4;table=2;up=0#sorted_table <font color="#ffffff">β-sheet (P)</font>]<font color="#ffffff"></font><br> <br />
<br />
|- class="twikiTableEven twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol" | <br />
d_αN<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 3.5<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3.4<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol3" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.2<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol4 twikiLastCol" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.2<br> <br />
<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted1 twikiTableRowdataBg1"<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol0 twikiFirstCol" | <br />
d_αN(i,i+2)<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 4.4<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol2" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3.8<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol3" | &nbsp; <br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol4 twikiLastCol" | &nbsp;<br />
|- class="twikiTableEven twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol" | <br />
d_αN(i,i+3)<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 3.4<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3.3<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol3" | &nbsp; <br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol4 twikiLastCol" | &nbsp;<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted1 twikiTableRowdataBg1"<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol0 twikiFirstCol" | <br />
d_αN(i,i+4)<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol1" | <br />
&nbsp;&nbsp; &nbsp; 4.2<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol2" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3.3<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol3" | &nbsp; <br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol4 twikiLastCol" | &nbsp;<br />
|- class="twikiTableEven twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol" | <br />
d_NN<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 2.8<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.6<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol3" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4.3<br> <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol4 twikiLastCol" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4.2<br> <br />
<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted1 twikiTableRowdataBg1"<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol0 twikiFirstCol twikiLast" | <br />
d_NN(i,i+2)<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol1 twikiLast" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 4.2<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol2 twikiLast" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4.1<br> <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol3 twikiLast" | &nbsp; <br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol4 twikiLastCol twikiLast" | &nbsp;<br />
|}<br />
<br />
For calibration of aromatic peaklists the characteristic distances are: <br />
<br />
<br> <br />
<br />
{| rules="rows" cellspacing="0" cellpadding="0" border="1" style="width: 285px; height: 106px;" id="table3" class="twikiTable"<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol" | <br> <br />
! valign="top" bgcolor="#687684" class="twikiTableCol1" | <br />
<font color="#ffffff">distance</font><font color="#ffffff"></font> <br />
<br />
! valign="top" bgcolor="#687684" class="twikiTableCol2 twikiLastCol" | <br />
<font color="#ffffff">center averaging</font><font color="#ffffff"></font> <br />
<br />
|- class="twikiTableEven twikiTableRowdataBgSorted1 twikiTableRowdataBg1"<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol0 twikiFirstCol" | <br />
d_δβ <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol1" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.33 - 3.77 <br />
<br />
| valign="top" bgcolor="#edf4f9" class="twikiTableCol2 twikiLastCol" | <br />
5.00 - 6.75 <br />
<br />
|- class="twikiTableOdd twikiTableRowdataBgSorted0 twikiTableRowdataBg0"<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol0 twikiFirstCol twikiLast" | <br />
d_δε <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol1 twikiLast" | <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.48 <br />
<br />
| valign="top" bgcolor="#ffffff" class="twikiTableCol2 twikiLastCol twikiLast" | <br />
&nbsp;&nbsp;&nbsp;&nbsp; 7.4 <br />
<br />
|}<br />
<br />
Note that aromatic QD and QE are pseudoatoms and the constraints in "center" averaging mode are much larger than the distances between individual atoms. <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
== '''References''' ==<br />
<br />
1.&nbsp;&nbsp; Wuthrich, K. (1986)&nbsp; "NMR of Proteins and Nucleic Acids", page 127. <br />
<br />
<br> <br />
<br />
<br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=GLOMSA&diff=3588
GLOMSA
2010-01-06T21:52:27Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
GLOMSA (GLObal Method for Stereospecific Assignment) is performed with the preliminary conformers and using all the local and global experimental data. If there are two NOEs of significantly different strength from a given proton to both diastereotopic substituents of a prochiral center and if the distances from the given proton to the two diastereotopic substituents differ consistently in the structures, the stronger NOE can be identified with the diastereotopic substituent that is closer to the given proton. <br />
<br />
[[Image:Glomsa1.jpg]] <br />
<br />
<br> <br />
<br />
If |Δb| &gt; cutoff, and |Δd| &gt; threshold, the diastereotopic pair is assigned as: <br />
<br />
**input (if Δb and Δd are both either positive or negative) <br />
**reversed (if Δb and Δd have opposite signs)<br><br> <br />
*GLOMSA usage: <br> <nowiki><br />
Parameters: atom selection (default: all atoms)<br />
cutoff=c (default: 0.4)<br />
threshold=t (default: 0.4)<br />
fraction=f (default: 100)<br />
</nowiki> <br> Only those atoms are considered, for which the difference between two constraints is larger than <tt>cutoff</tt> (in Å) and the corresponding average distance difference in the bundle of structure is larger than <tt>threshold</tt>. The minimal percentage of structures in which the sign of the distance difference must be consistent must be larger than <tt>fraction</tt> (in percent).<br />
<br />
== '''Running GLOMSA''' ==<br />
<br />
#Copy the latest manual refinement directory. <br />
#Download the sample [[Media:Run_glomsa.cya|run_glomsa.cya]] script <br />
#Start CYANA 2.1 on a workstation and execute <tt>run_glomsa.cya</tt>. This will generate <tt>glomsa.out</tt> file. <br />
#Analyze the output (here <tt>glomsa.out</tt>). For each stereospecific assignments verify that it is justified. Add confirmed assignments to the <tt>stereofound.cya</tt> file. <br />
#Run a manual structure calculation with the same <tt>CALC.cya</tt> as before. It will now use updated stereospecific assignments. Delete the <tt>glomsa.out</tt> file if you don't want it to be appended. <br />
#Repeat steps 3-5 until no new stereospecific assignments can be made.<br />
<br />
Important notes: <br />
<br />
*GLOMSA requires that you load a structure and UPL constraints. You should also declare the previously established stereospecific assignments (e.g., for VAL and LEU methyl groups) so that these could be used to determine new tereospecific assignments.<br> <br />
*'''Important!''' The UPL constraints supplied to GLOMSA must generated before distance modification with <tt>distance modify</tt>. When you follow the procedure in the [[ManualCYANARun|manual structure calculation]] example this would be the <tt>$name-in.upl</tt> file. <br />
*At this point we usually assign only HB2/3 spins and HA2/3 spins of Gly, HD21/22 of Asn and HE21/22 of Gln. Methyl QG1/2 of Val and QD1/2 of Leu can be assigned at this stage if that has not been done previously with the help of a fractionally <sup>13</sup>C-labeled sample. Stereospecific assignments of other spin pair are usually not reliable. <br />
*In the first iteration accept only stereospecific assignments supported by multiple third atoms.<br />
<br />
=== '''Acceptance criteria for stereospecific assignments from GLOMSA''' ===<br />
<br />
1.&nbsp; No conflicting stereospecific assignments in the output of GLOMSA (for example, both <tt>as input</tt> and <tt>reversed</tt> assignments for the same atom pair).&nbsp; <br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; Exceptions: <br />
<br />
*Some conflicting results may be caused by NOE peaks to QD and QE pseudoatoms of aromatic rings, due to constituent HD1/2 and HE1/2 atoms being so far away from the pseudoatom and slow ring flipping. <br />
*Some conflicting results may be caused by incorrect peak intensities (see next point).<br />
<br />
2.&nbsp; Verify that the NOE peak intensities (a-b1 and a-b2) are correct, and not biased by overlap with other peaks. <br />
<br />
3.&nbsp; Verify that the NOE peak intensities have not been misinterpreted (Example: HN-HB2 peak only resolved in <tt>n.peaks</tt>, and HN-HB3 - only in ali.peaks. Different NOE calibration constants for both peaklists may lead to incorrect stereospecific assignments). <br />
<br />
4.&nbsp; Big violations should be detected if the opposite stereospecific assignment is used. <br />
<br />
<br> <br />
<br />
=== '''run_glomsa.cya''' ===<br />
<br />
Here is an example script run_glomsa.cya: <br />
<pre>protocol:=glomsa.out<br />
read pdb $name.pdb<br />
read upl $name-in.upl<br />
stereofound # ssa from previous step<br />
atom glomsa<br />
protocol:=<br />
</pre> <br />
=== '''GLOMSA output''' ===<br />
<br />
The output of glomsa will look like this: <br />
<pre> Atoms residue constraints as input reversed distances assign<br />
sum min sum min num mean assign<br />
HB2/3 ARG 4<br />
H ARG 4 5.50 3.64 0.64 0.00 0.13 0.00 -15 -0.49 &nbsp;?<br />
HB2/3 LEU 6<br />
HA LEU 6 5.50 3.36 0.00 0.00 0.00 0.00 16 0.24 &nbsp;?<br />
QD1/2 LEU 6<br />
HA LEU 6 5.22 4.44 0.00 0.00 0.63 0.00 12 0.38 &nbsp;?<br />
HG12/3 ILE 8<br />
HA ILE 8 4.57 5.31 0.00 0.00 0.00 0.00 -10 0.00 &nbsp;?<br />
HB ILE 8 4.29 3.52 0.00 0.00 0.00 0.00 18 0.34 &nbsp;?<br />
HB2/3 LEU 9<br />
H VAL 10 3.76 4.38 5.27 0.00 0.00 0.00 20 1.16 reversed<br />
QD1/2 LEU 9<br />
HA LEU 9 5.56 3.85 0.05 0.00 7.15 0.00 18 0.51 &nbsp;?<br />
QG1/2 VAL 10<br />
H VAL 10 4.91 4.13 0.00 0.00 0.32 0.00 20 1.06 as input<br />
HA VAL 10 3.70 4.72 0.00 0.00 1.10 0.00 -20 -0.90 as input<br />
H CYS 11 4.97 5.87 0.00 0.00 0.00 0.00 -13 -0.10 &nbsp;?</pre> <br />
<br> For every spin pair there are lines listing spins. NMR cross-peaks to these spins have been used in an attempt to determine stereospecific assignments. <br />
<br />
<br> <br />
<br />
<br />
<br />
*[[Media:Run_glomsa.cya|run_glomsa.cya]]: CYANA script to run GLOMSA</div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=TALOS&diff=3587
TALOS
2010-01-06T21:50:02Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
TALOS (Torsion Angle Likelihood Obtained from Shift and sequence similarity) is a database system for empirical prediction of <tt>phi</tt> and <tt>psi</tt> backbone torsion angles from five kinds (HA, CA, CB, CO, N) of chemical shifts for a given protein sequence [1].&nbsp; In 2009, the Bax laboratory released a new and improved version of the program called TALOS+ [2].<br> <br />
<br />
For detailed information please check the [http://spin.niddk.nih.gov/NMRPipe/talos/ TALOS] and [http://spin.niddk.nih.gov/bax/software/TALOS+/index.html TALOS+] web sites.&nbsp; For installation questions and other support, you can also e-mail [mailto:shenyang@niddk.nih.gov Yang Shen]. <br />
<br />
== '''Using TALOS with CYANA''' ==<br />
<br />
=== '''Protocol at UB''' ===<br />
<br />
#Create a subdirectory (for example, <tt>structure/cyana21/talos</tt>) and copy the latest sequence and atom list files there. It is convenient to have them named <tt>XXXX.seq</tt> and <tt>XXXX.prot</tt>, where <tt>XXXX</tt> is an NESG target ID or other protein name. When using CARA, export the chemical shifts as an atom list file in this directory. <br />
#Create and init.cya in this directory as described in "[[NESG:CYANAInitFile|Creating an init.cya file for CYANA 2.1]]" or copy a previously used file. <br />
#Start CYANA and type:<br />
<br />
read prot XXXX.prot<br />
taloslist XXXX<br />
<br />
#This will create the TALOS input file <tt>XXXX.tab</tt>. In a UNIX shell run <tt>talos.tcl -in XXXX.tab</tt>. During the database search, a series of files <tt>pred/res*.tab</tt> will be created. Each one of these files tallies the 10 best database matches for a given residue in the target protein. Before exiting, <tt>talos.tcl</tt> calls <tt>vina.tcl</tt> to create a file <tt>pred.tab</tt>, which includes an initial summary of the prediction results. The database search will typically take about 10-60 sec per residue in the target. <br />
#In a UNIX shell run <tt>rama.tcl -in XXXX.tab</tt>. Here you can examine <tt>phi</tt> and <tt>psi</tt> distributions, choose database matches to be used in calculating predictions, and classify prediction results as <tt>Good</tt>, <tt>Ambiguous</tt> or <tt>Unclassified</tt> / <tt>New</tt>. See below for the guidelines for classifying prediction. Save your modifications in a new file, for example, <tt>talos.tab</tt>. <br />
#Start CYANA and type:<br />
<br />
talosaco pred #or "talos.tab" -- use the appropriate filename<br />
write aco talos.aco<br />
<br />
See also the <tt>~/demo/details/TalosAngleRestraints.cya</tt> example script in your local CYANA 2.1 installation. <br />
<br />
<br> <br />
<br />
=== '''Manual Refinement of TALOS Predictions''' ===<br />
<br />
Guidelines for refining the TALOS output: <br />
<br />
*Classify prediction as <tt>Good</tt> only if <br />
**All 10 best database matches fall in a "consistent" region of the Ramachandran map <br />
**Or 9 out of 10 best database matches fall in a consistent region with <tt>phi &lt; 0</tt>, and the one outlier also lies in <tt>phi &lt; 0</tt> half of the map <br />
**Or 9 out of 10 of the best database matches fall in a consistent region with <tt>phi &gt; 0</tt> <br />
*Accept predictions which are classified as <tt>Good</tt>, whose residues are in beta-sheets or helices according to CSI (excluding the first and the last residue of a secondary structure element).<br />
<br />
For ''de novo'' structure determination it is recommended to take the automatically generated TALOS constraints. Angular constraints outside of secondary structure elements (as determined by CSI) can be commented out in the <tt>talos.aco</tt> file. <br />
<br />
During structure refinement you can refine TALOS predictions against a preliminary structure. <br />
<pre>vina.tcl -in XXXX.tab -ref XXXX.pdb -auto</pre> <br />
and <br />
<pre>rama.tcl -in XXXX.tab -ref XXXX.pdb</pre> <br />
<br> The <tt>XXXX.pdb</tt> file '''must''' have only one conformer. Thus, you may need to analyze the angle distributions in a molecular graphics package (e.g. MOLMOL). <br />
<br />
<br> <br />
<br />
{| border="1"<br />
|-<br />
| Element <br />
| PHI <br />
| PSI<br />
|-<br />
| α-helix <br />
| -60 <br />
| -45<br />
|-<br />
| β-sheet <br />
| -140 <br />
| 135<br />
|}<br />
<br />
<br> <br />
<br />
=== '''Description of talosaco.cya Macro''' ===<br />
<br />
Invoked as: <br />
<pre>talosaco file [factor [width]]</pre> <br />
Here <tt>file</tt> is the TALOS prediction output, <tt>width</tt> is the threshold minimum width for <tt>PHI/PSI</tt> angle distributions, and <tt>factor</tt> is used to scale the width of a distribution when creating an angle constraint. Both <tt>width</tt> and <tt>factor</tt> arguments are optional. By default, <tt>width=20.0</tt> and <tt>factor=2.0</tt>. <br />
<br />
This macro will create angle constraints for a given residue only if the prediction is classified as "Good" and the residue is not a proline. <br />
<br />
<br> <br />
<br />
== '''Using TALOS and TALOS+ at CABM''' ==<br />
<br />
=== Preparing for a TALOS+ run ===<br />
<br />
*Make a sub-directory in your project for TALOS. <br />
*you will need the following files in your directory: <br />
*a bmrb file in 2.1 format. &nbsp;Here is an [[Media:PfR193A_062509_2.1f_4CYANA.bmrb|example]]. <br />
*[[Media:BMRBParsing.pm|BMRBParsing.pm]]:&nbsp; BMRB&nbsp;parser <br />
*[[Media:Tab4Talos.pl|Tab4Talos.pl]]:&nbsp; perl script to prepare input file for TALOS <br />
*[[Media:Talos2dyana_taloserrors.pl|talos2dyana_taloserrors.pl]]:&nbsp; perl script to prepare a CYANA .aco file <br />
*Run the following command:<br />
<pre> Tab4Talos.pl [.bmrbf] [input4Talos]<br />
<br />
</pre> <br />
This make an input chemical shift list for TALOS.&nbsp; Here is an [[Media:PfR193A_4Talos.input|example]].<br> <br />
<br />
=== Running TALOS+ and making a dihedral angle constraint file<br> ===<br />
<br />
*Next run talos+:<br />
<pre> talos+ -in [input4Talos</pre> <br />
This makes a number of output files including the pred.tab.&nbsp; <br> <br />
<br />
*Next, edit the pred.tab and comment out (#) any lines that do not have the "10 Good" comment.&nbsp; <br />
*Finally, run the talos2cyana perl script to make a CYANA .aco file with only the results classified as "10 Good", and using the phi and psi errors given by TALOS.&nbsp; They user can modify this script to make his/her own error limits (i.e., +/- 20 or 30).<br />
<pre> perl talos2dyana_taloserrors.pl pred.tab [output.aco]<br />
</pre> <br />
<br> <br />
<br />
== '''References''' ==<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10212987?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=3 <font face="Arial,Helvetica">1.&nbsp;&nbsp;&nbsp; Cornilescu, G., Delaglio, F. and Bax, A. (1999) </font><font face="Arial,Helvetica">Protein backbone angle restraints from searching a database for chemical shift and sequence homology. </font><font face="Arial,Helvetica">''J. Biomol. NMR 13'', 289-302.</font>] <br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19548092?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1 <font face="Arial,Helvetica">2.&nbsp;&nbsp;&nbsp; Shen, Y., </font><font face="Arial,Helvetica">Delaglio, F., Cornilescu, G. and Bax, A. (2009) </font><font face="Arial,Helvetica">TALOS+: A hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. </font><font face="Arial,Helvetica">''J. Biomol. NMR&nbsp;44'', 213-223</font>] <br />
<br />
<br> <br />
<br />
<br><br></div>
Jma
https://nesgwiki.chem.buffalo.edu/index.php?title=FOUND&diff=3586
FOUND
2010-01-06T21:48:05Z
<p>Jma: </p>
<hr />
<div>== '''Introduction''' ==<br />
<br />
'''FOUND''' performs systematic local conformation analysis by exhaustive grid searches for arbitrary contiguous fragments of proteins and nucleic acids. It uses torsion angles as the only degrees of freedom to identify all conformations that fulfill the steric and NMR-derived conformational restraints within a contiguous molecular fragment, as defined either by limits on the maximal restraint violations or by the fragment-based target function value. <br />
<br />
'''FOUND''' does the following: <br />
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*Detects inconsistencies in the experimental. <br />
*Derives stereospecific assignments of diastereotopic methylene protons or isopropyl groups. <br />
*Converts coupling constants and UPLs into torsion angle restraints.<br />
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Parameters:&nbsp; <br />
<pre> range=residue range (default: all amino acid residues)<br />
angles=side-chain angles (default: CHI1)<br />
tfcut=fmax (default: 0.0)<br />
continue</pre> <br />
<br> Performs for all amino acid residues in the given range grid searches comprising the backbone dihedral angles φ, ψ, χ1 and the given side-chain angles. To specify more than one side-chain angle, the names must be given, separated by blanks and enclosed in double quotes. If the cutoff value for the local, fragment-based target function, <tt>fmax</tt>, is positive, then all conformations with a local target function value below <tt>fmax</tt> will be considered as allowed. Otherwise, a conformation will be allowed if no single restraint violation exceeds the corresponding cutoff value defined by the variables <tt>soft_upl</tt>, <tt>soft_lol</tt>, etc. Unless the <tt>continue</tt> option is set, the allowed ranges of dihedral angles will be initialized to allow all possible angle values before the grid searches are started. <br />
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FOUND is implemented as a CYANA and DYANA macro succeeding the standalone HABAS program. Therefore, the FOUND macros are called <tt>habas.cya</tt> or <tt>habas.dya</tt>. For more information type <tt>help habas</tt> in CYANA or see the reference below. <br />
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References:<br> FOUND - [http://www.springerlink.com/content/t318k12296032kk5/fulltext.pdf Guntert P. ''J. Biomol. NMR'' 12 (4): 543-548 NOV 1998]<br> HABAS - [http://pubs.acs.org/cgi-bin/archive.cgi/jacsat/1989/111/i11/pdf/ja00193a036.pdf Guntert P. ''J. Am. Chem. Soc.'' 1989, 111, 3997-4004] <br />
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== '''How FOUND (HABAS) Works'''<br> ==<br />
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[[Image:Found3.jpg]]<br> <br />
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== '''Preparing to run FOUND (HABAS)''' ==<br />
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If you are using completely unassigned peaklists and you don't have any J-coupling data, then you don't need to run found. Otherwise, do the following: <br />
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#Create a working subdirectory (for example, <tt>structure/cyana21/found</tt>). <br />
#If you have ct-[<sup>13</sup>C,<sup>1</sup>H]-HSQC spectra for the fractionally <sup>13</sup>C-labeled sample, determine the stereospecific assignments for Val and Leu methyl groups and swap spin labels (in the XEASY atom list or CARA repository) where necessary, as described in [[NESG:SSAFromFractional13CLabeledSample|Stereospecific assignments from fractionally 13C-labeled sample]]. Create a file <tt>stereo.cya</tt> to declare the residues, for which stereospecific assignments were established. <br />
#Copy the latest sequence (<tt>XXXX.seq</tt>), atomlist (<tt>XXXX.prot</tt>) and peaklist files (<tt>n.peaks</tt>, <tt>ali.peaks</tt> and <tt>aro.peaks</tt>) into the working directory. Modify the sequence file if you have non-standard residues, such as cis-proline (use <tt>cPRO</tt>). <br />
#Create an init.cya file as described in "[[NESG:CYANAInitFile|Creating an init.cya file for CYANA 2.1]]" or copy a previously used file. Set an appropriate RMSD calculation range. <br />
#Create a <tt>GridSearch.cya</tt> file or download a template [[Media:GridSearch.cya|GridSearch.cya]] (see below). <br />
#Copy the TALOS dihedral angel constraints file (<tt>talos.aco</tt>) from the previous step. Keep only those constraints, which correspond to residues in the secondary structure elements according to CSI, excluding one residue at the edge of each element. You can comment out the remaining lines with a hash character =#= using any text editor. <br />
#Produce initial calibration as described in NOECalibrationCYANA#Finding_Initial_NOE_Calibration. <br />
#Download the [[Media:CALC_found.cya|CALC.cya]] file to run a test structure calculation using the output of FOUND (see below).<br />
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Here is the template <tt>GridSearch.cya</tt> script adapted from <tt>~/demos/details/GridSearch.cya</tt> in CYANA 2.1 installation directory. The options for scalar couplings have been commented out since they aren't routinely used. <br />
<pre>protocol:=found.out<br />
read seq $name.seq<br />
read upl short.upl # read noe upper distance limits<br />
stereo # apply stereospecific assignments from stereo.cya<br />
#read cco demo.cco # read J-coupling constants<br />
#karplus # use standard Karplus curves (from karplus.cya)<br />
#habas angles="CHI1" tfcut=0.05 # perform phi/psi/chi1 grid search (HABAS)<br />
habas angles="CHI1 CHI2*" tfcut=0.05 # alternative phi/psi/chi1/chi2 grid search<br />
gridplot habas.ps<br />
atom stereo list<br />
write upl swapped.upl # write upper limits with swapped stereo pairs<br />
write aco gridsearch.aco # write upper limits with swapped stereo pairs<br />
#write cco swapped.cco # write couplings with swapped stereo pairs<br />
savestereo stereofound.cya # write macro with stereospecific assignments<br />
protocol:=<br />
</pre> <br />
<br> It is recommended to apply [[NESG:SSAFromFractional13CLabeledSample|stereospecific assignments of Leu and Val isopropyl groups]] (if available) when running HABAS. This may help HABAS derive additional stereospecific assignments for HB2/3 spins of Leu. To take advantage of stereospecific assignments create <tt>swap.cya</tt> and <tt>stereo.cya</tt> macros as described in the topic about [[NESG:ApplyingSSA|applying stereospecific assignments]] and use them to generate a swapped proton list and a swapped UPL file <tt>short.upl</tt> <br />
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'''N.B.''' The <tt>GridSearch.cya</tt> script is not the same as the <tt>grid search</tt> command! <br />
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== '''Verifying Local NOE Network''' ==<br />
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#In CYANA 2.1 run the <tt>GridSearch.cya</tt> script. <br />
#In CYANA 2.1 run the <tt>CALC.cya</tt> script. This may take several minutes on a workstation. <br />
#Go residue-by-residue through the FOUND output. If inconsistent data was found, double-check the NOE peaks for this residue in XEASY or CARA. UPL violations reported in the <tt>XXXX.ovw</tt> file usually help to pinpoint the questionable peaks.<br />
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**Usually ''inconsistent data'' reports are caused by peaks with improper volumes or incorrectly assigned peaks. Check assignments and integration in XEASY or CARA. <br />
**Too many ''inconsistent data'' reports and violations for apparently valid peaks indicate that calibration that is too tight. Look at the violations in the <tt>XXXX.ovw</tt> file to determine which peaklist requires calibration adjustment. Change calibration constants and re-run the calibration.<br />
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#Repeat steps 1 and 3 until no inconsistent data was found by FOUND and the average target function is &lt; 0.5 .<br />
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FOUND will produce the following files: <br />
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*<tt>stereofound.cya</tt> - CYANA script, which delcares stereospecific assignments. It is a combination of input stereospecific assignments and those generated by FOUND. <br />
*<tt>swapped.upl</tt> - a modification of the input UPL file, in which spin labels have been swapped according to the newly derived stereospecific assignments. This file itself is not used in subsequent automated structure calculation and NOE assignment, because of incompatible NOE calibration protocols. <br />
*<tt>gridsearch.aco</tt> - is a combination of input TALOS constraints and constraints generated with the grid search method.<br />
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== '''Stereospecific Assignments from FOUND (HABAS)''' ==<br />
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Stereo-specific assignment can be obtain from checking FOUND module without any preliminary structure. FOUND module performing separate grid searches for both possible stereospecific assignments for diastereotopic protons or isopropyl groups as show in the figure below:<br> <br />
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[[Image:Found6.jpg]] <br />
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where the allowed conformation numbers for the two possible stereospecific assignments are N1 and N2, separately <br />
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*if N1 &gt;0, N2 &gt;0, stereospecific assignment is ambiguous <br />
*if N1 &gt;0, N2 =0, assigned as input <br />
*if N1 =0, N2 &gt;0, assigned as reversed <br />
*if N1 =0, N2 =0, both assignment possibilities are disallowed - inconsistent data.<br />
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<br> After running FOUND module check by using CYANA macro "GridSeach.cya" check the output file (e.g. see figure below) for stereospecific assignments. This fragment shows that stereospecific assignments were derived for HB2/3 of Arg33 and Met45. Note that externally declared stereospecific assignments, such as QD1/2 of Leu34 and QG1/2 of Val38 in this example, are also listed.&nbsp; <br />
<pre> 33 ARG HB2 HB3 assigned<br />
33 ARG HG2 HG3<br />
33 ARG HD2 HD3<br />
33 ARG HH11 HH12<br />
33 ARG HH21 HH22<br />
34 LEU HB2 HB3<br />
34 LEU QD1 QD2 assigned<br />
36 PHE HB2 HB3<br />
37 PRO HB2 HB3<br />
37 PRO HG2 HG3<br />
37 PRO HD2 HD3<br />
38 VAL QG1 QG2 assigned<br />
45 MET HB2 HB3 assigned<br />
45 MET HG2 HG3<br />
</pre> <br />
See the data upstream from the summary to find whether atom pair were assigned "swapped" or "as input". Verify that peaks assigned to the reported spins are integrated correctly. Avoid accepting assignments with very few allowed conformations (such as Met45 below), because they are indicative of too tight UPLs.&nbsp; <br />
<pre> ARG 33:<br />
HB2/HB3 ARG 33: 0/1465 conformations, assigned reversed, swapped.<br />
HG2/HG3 ARG 33: 698/767 conformations, unassigned.<br />
...<br />
MET 45:<br />
HB2/HB3 MET 45: 35/0 conformations, assigned as input.<br />
HG2/HG3 MET 45: 14/21 conformations, unassigned.<br />
</pre> <br />
Delete or comment out those lines in <tt>stereofound.cya</tt>, which you think correspond to incorrectly derived stereospecific assignments. <br />
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In subsequent steps it is recommended to swap the labels in the original source containing chemical shift assignments: the atom list if using XEASY, or CARA repository if using CARA. <br />
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<br> <br />
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*[[Media:GridSearch.cya|GridSearch.cya]]: CYANA script to run FOUND module<br />
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*[[Media:CALC_found.cya|CALC.cya]]: CYANA script to run manual calculation with local constraints</div>
Jma