NESG Wiki - User contributions [en]

Main Page

2018-11-02T16:47:47Z

Alex:

__NOTOC__

<big>'''Welcome to the NESG Wiki!'''</big> 

The NESG Wiki is a medium for sharing experimental protocols as well as training an educational materials in the fields of structural biology, structural genomics and biomolecular NMR.

Please check out [[NESG NMR wiki workshop at the 2010 Keystone meeting|NESG NMR wiki workshop presentations at the 2010 Keystone meeting]]

== Protein Sample Production ==

{| cellspacing="1" class="FCK__ShowTableBorders"
|- valign="top"
|
*[[Target selection|NESG target selection]] 
*[[DNA cloning protocols|DNA cloning protocols]] 
*[[Protein purification|Protein expression and purification protocols]]  
*Sample Optimization
**[[Construct optimization]]
**[[Buffer optimization]]
**[[Cofactor optimization]]

|
*Protein Sample Analysis
**[[SDS page gel]]
**[[Protein concentration|Protein concentration measurements]]
**[[Oligomerization Status|Assessment of Oligomerization State]]
***[[Gel filtration and light scattering|gel-filtration and light scattering]]
***[[NMR determined Rotational correlation time]]
**[[MassSpectrometry|Mass spectrum]]
**[[NMR screening]]

 

|}

== NMR Data Acquisition ==

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
*Routine operation
**[[NMR Sample Preparation]]
**[[Inserting NMR Sample]]
**[[Tuning and matching]]
**[[Deuterium Lock]]
**[[Shimming]]
**[[Pulse width calibration]]
**[[Temperature calibration]]
**[[Chemical shift referencing]]
*Advanced operation
**[[Deuterium pulse width calibration and decoupling]]

|
*NMR data acquisition for protein structure determination
**[[Common NMR experiment sets]]
**[[NMR experiment setup scripts for VNMRJ|Custom NMR experiment setup scripts for VNMRJ]]
**[[Estimation of rotational correlation time]]
**[[Estimation of measurement time]]
**[[Simultaneous 13C,15N-resolved NOESY]]
**[[2D (13C, 1H) HSQC for fractionally 13C-labeled samples|2D [13C, 1H]-HSQC for fractionally 13C-labeled samples]]
**[[Long-range 15N-1H correlation experiments for histidine rings]]
*[[Setting up non-uniformly sampled spectra|Non-uniform sampling (NUS) ]]
**[[Setting up non-uniformly sampled spectra/NUS guide for Varian|NUS - Varian]]
**[[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|NUS - Bruker]]

|
*Maintenance (VARIAN)
**[[Installing and updating BioPack]]
**[[Full probefile calibration]]
**[[Rebooting spectrometer console]]
**[[Conditioning procedure for cryogenic probes]]

|}

== NMR Data Processing ==

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
NMRPipe

*[[Brief description of philosophy, commands, and functions of NMRPipe|Brief description of philosophy, commands, and functions]]
*[[Routine 2D Experiment|2D experiments]]
*[[Routine Processing Procedure for 3D 15N and 13C-edited Experiments|3D 15N and 13C-edited experiments]]
*[[HSQCTROSY RDC Measurement|2D ]][[HSQCTROSY RDC Measurement|15]][[HSQCTROSY RDC Measurement|N HSQC-TROSY experiment for RDC measurement]]
*[[Jmodulation Experiment RDC|2D J-modulation experiment for RDC measurement]]
*[[Alignment Media Preparation|Alignment Media Preparation for RDC measurement]]

|
Other

*[[Processing NMR spectra with PROSA|PROSA]]
*[[Bruker Data Processing|TOPSPIN]]
*[[AGNuS/AutoProc|AUTOPROC]]
*[[Processing non-uniformly sampled spectra with Multidimensional Decomposition|Processing NUS spectra with MDD]]
*[[Spectra Format Conversion from NMRPipe Data|NMRPipe processed data conversion to Sparky, CARA, XEASY, and NMRViewJ]]

|}

== NMR Resonance Assignment ==

*[[Resonance Assignment/Principles and concepts|Principles and concepts]]

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
*Semi-automated protocols
**[[Resonance Assignment/CARA|CARA]]
**[[Sparky]]
**[[Resonance Assignment/XEASY|XEASY]]

|
*Automated resonance assignment
**[[AutoAssign|AutoAssign]]
**[[AutoAssign WebServer|AutoAssign server]]
**[[Abacus|ABACUS]]
**[[The PINE Server|PINE server]]

|
*Validation and deposition
**[[AVS|Assignment validation suite (AVS)]]
**[[LACS|Linear analysis of chemical shift (LACS)]]
**[[PDB and BMRB Deposition#Preparing_files_for_BMRB_depostion|Depositing chemical shifts]]

|}

== Structure Calculation and Validation ==

[[Structure Calculation and Validation|Principles and concepts]]

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
*Structure calculation
**[[CYANA Structure Determination Program|CYANA]]
**[[AutoStructure Structure Determination Program|AutoStructure]]
**[[Structure Calculation Using CS-Rosetta|CS-ROSETTA]]
**[[Structure Calculation Using CS-DP ROSETTA|CS-DP ROSETTA]]
**[[Structure Calculation Using CS-RDC-ROSETTA|CS-RDC-ROSETTA]]
**[[Consensus Approaches|"Consensus" approaches]]
**[[Refinement Strategies|Refinement Strategies]]

|
*Special topics
**[[Protein-Ligand Complexes|Protein-Ligand complexes]]
**[[Working With Metal Ions|Metal ions]]
**[[Residual Dipolar Couplings in Structure Refinement|Residual Dipolar Couplings]]
**[[REDCAT|REDCAT]] and [[REDCRAFT|REDCRAFT]]
**[[Paramagnetic Constraints in Structure Determination|Paramagnetic constraints]]
**[[RDC-Assisted Dimer Structure Determination|RDC-assisted dimer structure calculation]]

|
*Structure Refinement
**[[Structure Refinement Using CNS Energy Minimization With Explicit Water|CNS refinement]]
**[[Structure Refinement Using XPLOR-NIH|XPLOR-NIH refinement]]
**[[Rosetta High Resolution Protein Structure Refinement Protocol|ROSETTA refinement]] 

|
*Validation and deposition
**[[PdbStat|PdbStat]]
**[[PSVS|PSVS]]
**[[RPF Analysis|RPF analysis]]
**[[MolProbity Server|MolProbity server]]
**[[RDCvis & KiNG|RDCvis]]
**[[PDB and BMRB Deposition|PDB and BMRB deposition]]
**[[ADIT-NMR|ADIT-NMR]]
**[[HarvestDB|HarvestDB]]
**[[SPINS|SPINS]]

|}

== nmr 2.0 ==

[[http://www.nmr2.buffalo.edu/ Homepage]]

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|

*[http://www.nmr2.buffalo.edu/comm/links/ nmr 2.0 Communication]
*[http://www.nmr2.buffalo.edu/resources/edu/ nmr 2.0 Educational]

*[http://www.nmr2.buffalo.edu/resources/jobs/ nmr 2.0 Jobs]
*[http://www.nmr2.buffalo.edu/resources/jobprofiles/ nmr 2.0 Job Seekers]

*[http://www.nmr2.buffalo.edu/resources/poll/ nmr 2.0 News]

*[http://www.nmr2.buffalo.edu/resources/queries/ nmr 2.0 Queries]

*[http://www.nmr2.buffalo.edu/resources/humor/ nmr 2.0 Humor]

*[http://www.nmr2.buffalo.edu/resources/links/ nmr 2.0 Links]

*[http://www.nmr2.buffalo.edu/blog nmr 2.0 Blog]

|}

For a more linear view of the contents including those in development see [[Wiki Tree Layout]]

Routine Processing Procedure for 3D 15N and 13C-edited Experiments

2017-11-16T21:57:51Z

Alex: Undo revision 4338 by Alex (Talk)

'''NMR Data Processing > Routine 3D Experiments via NMRPipe'''
<div> </div>
===== Brief Description =====

The procedure for processing a 3D dataset is fairly similar to the ones described for the 2D dataset along with additional parameters for a third dimension (generally refered to as the z dimension). For nD dataset where the indirect dimension(s) is collected with few points, it is often advantageous to increase the number of points and digital resolution using ‘Linear Prediction’. This calculation determines the frequency and decay rate of the peaks in an FID or interferrogram, and extends them mathematically. It is very useful for 15N and 13C-edited 3D dataset where experimental time is needed to improve S/N, so the number of increments is limited. It is important to mention that ‘Linear Prediction’ on the selected dimension of a 3D dataset is performed after the other two dimensions have been processed. An example processing script for HNCO experiment is shown here, where the HN dimension (x) and N dimension (z) are first processed followed by the linear prediction and processing of the CO dimension (y). The N dimension is then inverse transformed, linear predicted, and retransformed. This processing script is also applicable to the majority of 3D 15N and 13C-edited datasets for protein related work.

===== Software Information =====

NMRPipe (download information and user manual) [http://spin.niddk.nih.gov/NMRPipe/ http://spin.niddk.nih.gov/NMRPipe/] Brief descriptions of specific functions are accessible via nmrPipe –

:‘nmrPipe –help’ will list most functions.
:‘nmrPipe –fn GM –help’ will give description of the GM function.

 Supported Platforms

:Linux (RedHat Linux/Fedora)
:Mac OS X (10.3.4 and up)
:SGI Irix  (6.2 and up)
:Sparc Solaris (2 and up)
:Windows XP Pro with Microsoft Services for UNIX (SFU 3.5)

===== Converting Spectrometer Data into NMRPipe Format =====

The conversion of data from spectrometer format to NMRPipe format is usually done using a build-in program called ‘varian’ for Varian a dataset ('bruker' for a Bruker dataset and 'delta' for dataset from JEOL). [[Brief description of philosophy, commands, and functions of NMRPipe|Brief summaries of the build-in program can be found here]]. 

 

Step 1: Under the same directory where the procpar and fid files are, type ‘varian’ and this will start a tcl/tk script that brings up two windows.

[[Image:Varian-utility-raw.png]] [[Image:Varian-script-raw.png]] 

Step 2: Click on the ‘Read Parameters’ button and the script will read the parameter file (‘procpar’) and update the parameters.

[[Image:Varian-utility-3D.png]] [[Image:Varian-script-3D.png]] 

The template shows parameters for three dimensions x, y, and z for HN, CO, and N, respectively. More detailed description of the input script can be found at routine 2D experiment or NMRPipe user manual.

:‘-aqORD 1’ !!! Varian dataset when ‘array=phase,phase2’
:‘-xMODE complex’ !!! For the direct dimension
:‘-yMODE: complex’ !!! For the CO dimension
:‘-zMODE: Rance-Kay’ !!! For sensitivity enhanced experiment

 

Step 3: Click on ‘Save Script’, and then ‘Execute Script’. The converted planes in NMRPipe format will be stored in the ‘data’ directory along with an UNIX shell script called ‘fid.com’. 

===== Processing and Visualizing 3D Dataset =====

For information on the macro editor of nmrDraw, see [[Routine 2D Experiment|routine processing procedure for 2D experiment]]. An example of processing script for a 3D HNCO spectrum with linear prediction in the y and z dimensions is shown below.
<pre>!!! Part 1, process the directly-detected x-axis !!!
xyz2pipe -in fid/test%03d.fid -x -verb \
| nmrPipe -fn SOL \ ## Removed residual solvent
| nmrPipe -fn SP -off 0.5 -end 0.98 -pow 2 -c 0.5 \ ## Apodization
| nmrPipe -fn ZF -auto \ ## Zero fill
| nmrPipe -fn FT \ ## Fourier transformation
| nmrPipe -fn PS -p0 43 -p1 0.0 -di \ ## Phase correction
| nmrPipe -fn EXT -left -sw \ ## Extract left half spectrum
| pipe2xyz -out lp/test%03d.ft3 -x

!!! Part 2, process the indirectly-detected z-axis !!!
xyz2pipe -in lp/test%03d.ft3 -z -verb \
| nmrPipe -fn SP -off 0.5 -end 0.95 -pow 1 -c 0.5 \ ## Apodization
| nmrPipe -fn ZF -auto \ ## Zero fill
| nmrPipe -fn FT \ ## Fourier transformation
| nmrPipe -fn PS -p0 0.0 -p1 0.0 -di \ ## Phase correction
| pipe2xyz -out lp/test%03d.ft3 -z –inPlace

!!! predict and process the indirect-detected y-axis !!!
xyz2pipe -in lp/test%03d.ft3 -y -verb \
| nmrPipe -fn LP -fb -ord 10 \ ## Linear Prediction
| nmrPipe -fn SP -off 0.5 -end 0.98 -pow 1 -c 1.0 \ ## Apodization
| nmrPipe -fn ZF -auto \ ## Zero fill
| nmrPipe -fn FT \ ## Fourier transformation
| nmrPipe -fn PS -p0 -135 -p1 180 -di \ # Phase correction
| pipe2xyz -out lp/test%03d.ft3 -y -inPlace

!!! inverse, predict, and re-process the z-axis
xyz2pipe -in lp/test%03d.ft3 -z -verb \
| nmrPipe -fn HT -auto \
| nmrPipe -fn PS -inv -hdr \
| nmrPipe -fn FT -inv \
| nmrPipe -fn ZF -inv \
| nmrPipe -fn SP -inv -hdr \
| nmrPipe -fn LP -fb \ ## Linear prediction
| nmrPipe -fn SP -off 0.5 -end 0.98 -pow 1 -c 0.5 \ ## Apodization
| nmrPipe -fn ZF -auto \ ## Zero fill
| nmrPipe -fn FT \ ## Fourier transformation
| nmrPipe -fn PS -hdr -di \
| pipe2xyz -out lp/test%03d.ft3 -z -inPlace
</pre>
===== Suggested Workflow =====

:Step 1: Generate the processing script with the appropriate commands and functions using the macro editor or modified existing script. Set both the p0 and p1 phasing values of the direct dimension to 0.
:Step 2: Execute the processing script in a UNIX terminal.  The 2D projection planes of the 3D dataset can be created using the build-in program readROI for phase correction  (execute the following command in a UNIX terminal, ‘nmrWish –f [[Media:Project.txt|Project.txt]]’).  Load the projection plane into nmrDraw to perform phase correction and optimize the applied functions. A 1D horizontal trace can be activate by typing ‘h’ in the spectrum window and the phase of the dimension can be adjusted using the P0 and P1 slider bars. To phase an indirect dimension, type 'v'.  Now the P0 and P1 slider bars will change the phase of the indirectly detected dimension.
:Step 3: Execute the processing script with optimized parameters.
:Step 4: Convert the processed data to the format of graphical NMR assignment program.

 Updated by Hsiau-Wei Lee, 2011

Main Page

2017-11-16T21:56:30Z

Alex: Reverted edits by Pwils3 (Talk) to last revision by Admin

__NOTOC__

<big>'''Welcome to the NESG Wiki!'''</big> 

The NESG Wiki is a medium for sharing experimental protocols as well as training an educational materials in the fields of structural biology, structural genomics and biomolecular NMR.

Please check out [[NESG NMR wiki workshop at the 2010 Keystone meeting|NESG NMR wiki workshop presentations at the 2010 Keystone meeting]]

== Protein Sample Production ==

{| cellspacing="1" class="FCK__ShowTableBorders"
|- valign="top"
|
*[https://jomadjeans.wordpress.com/ Target] selection|NESG target selection 
*[[DNA cloning protocols|DNA cloning protocols]] 
*[https://www.quora.com/unanswered/How-comfortable-are-JOMAD-Jeans Pro]tein purification|Protein expression and purification protocols  
*Sample Optimization
**[[Construct optimization]]
**[[Buffer optimization]]
**[[Cofactor optimization]]

|
*Protein Sample Analysis
**[[SDS page gel]]
**[[Protein concentration|Protein concentration measurements]]
**[[Oligomerization Status|Assessment of Oligomerization State]]
***[[Gel filtration and light scattering|gel-filtration and light scattering]]
***[[NMR determined Rotational correlation time]]
**[[MassSpectrometry|Mass spectrum]]
**[[NMR screening]]

 

|}

== NMR Data Acquisition ==

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
*Routine operation
**[[NMR Sample Preparation]]
**[[Inserting NMR Sample]]
**[[Tuning and matching]]
**[[Deuterium Lock]]
**[[Shimming]]
**[[Pulse width calibration]]
**[[Temperature calibration]]
**[[Chemical shift referencing]]
*Advanced operation
**[[Deuterium pulse width calibration and decoupling]]

|
*NMR data acquisition for protein structure determination
**[[Common NMR experiment sets]]
**[[NMR experiment setup scripts for VNMRJ|Custom NMR experiment setup scripts for VNMRJ]]
**[[Estimation of rotational correlation time]]
**[https://www.jomadjeans.com High waisted flare jeans]
**[[Simultaneous 13C,15N-resolved NOESY]]
**[[2D (13C, 1H) HSQC for fractionally 13C-labeled samples|2D [13C, 1H]-HSQC for fractionally 13C-labeled samples]]
**[https://www.jomadjeans.com Ankle grazer jeans]
*[[Setting up non-uniformly sampled spectra|Non-uniform sampling (NUS) ]]
**[https://www.jomadjeans.com designer jeans for women]
**[[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|NUS - Bruker]]

|
*Maintenance (VARIAN)
**[[Installing and updating BioPack]]
**[http://prime.ece.mtu.edu/wiki/index.php?title=User:Jomadjeans Full probefile calibration]
**[http://jomadjeansnyc.wikidot.com/ Reboot]ing spectrometer console
**[http://jomadjeansdesignerjeansforwomen.wikidot.com Conditioning procedure for cryogenic probes]

|}

== NMR Data Processing ==

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
NMRPipe

*[[Brief description of philosophy, commands, and functions of NMRPipe|Brief description of philosophy, commands, and functions]]
*[[Routine 2D Experiment|2D experiments]]
*[[Routine Processing Procedure for 3D 15N and 13C-edited Experiments|3D 15N and 13C-edited experiments]]
*[https://rustednail56.wixsite.com/jomadjeans HSQCTROSY] RDC Measurement|2D ]][[HSQCTROSY RDC Measurement|15]][[HSQCTROSY RDC Measurement|N HSQC-TROSY experiment for RDC measurement
*[https://rustednail56.wixsite.com/jomadjeansforwomen Jmodulation] Experiment RDC|2D J-modulation experiment for RDC measurement
*[https://www.kiwibox.com/JOMAD_Jeans/ Alignment] Media Preparation|Alignment Media Preparation for RDC measurement

|
Other

*[[Processing NMR spectra with PROSA|PROSA]]
*[[Bruker Data Processing|TOPSPIN]]
*[https://www.jomadjeans.com/collection/all Fall Jeans]
*[[Processing non-uniformly sampled spectra with Multidimensional Decomposition|Processing NUS spectra with MDD]]
*[https://www.jomadjeans.com/collection/all Spring Jeans]

|}

== NMR Resonance Assignment ==

*[[Resonance Assignment/Principles and concepts|Principles and concepts]]

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
*Semi-automated protocols
**[[Resonance Assignment/CARA|CARA]]
**[[Sparky]]
**[[Resonance Assignment/XEASY|XEASY]]

|
*Automated resonance assignment
**[[AutoAssign|AutoAssign]]
**[[AutoAssign WebServer|AutoAssign server]]
**[[Abacus|ABACUS]]
**[[The PINE Server|PINE server]]

|
*Validation and deposition
**[[AVS|Assignment validation suite (AVS)]]
**[[LACS|Linear analysis of chemical shift (LACS)]]
**[[PDB and BMRB Deposition#Preparing_files_for_BMRB_depostion|Depositing chemical shifts]]

|}

== Structure Calculation and Validation ==

[[Structure Calculation and Validation|Principles and concepts]]

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
*Structure calculation
**[[CYANA Structure Determination Program|CYANA]]
**[[AutoStructure Structure Determination Program|AutoStructure]]
**[[Structure Calculation Using CS-Rosetta|CS-ROSETTA]]
**[[Structure Calculation Using CS-DP ROSETTA|CS-DP ROSETTA]]
**[[Structure Calculation Using CS-RDC-ROSETTA|CS-RDC-ROSETTA]]
**[[Consensus Approaches|"Consensus" approaches]]
**[[Refinement Strategies|Refinement Strategies]]

|
*Special topics
**[[Protein-Ligand Complexes|Protein-Ligand complexes]]
**[[Working With Metal Ions|Metal ions]]
**[[Residual Dipolar Couplings in Structure Refinement|Residual Dipolar Couplings]]
**[[REDCAT|REDCAT]] and [[REDCRAFT|REDCRAFT]]
**[[Paramagnetic Constraints in Structure Determination|Paramagnetic constraints]]
**[[RDC-Assisted Dimer Structure Determination|RDC-assisted dimer structure calculation]]

|
*Structure Refinement
**[[Structure Refinement Using CNS Energy Minimization With Explicit Water|CNS refinement]]
**[[Structure Refinement Using XPLOR-NIH|XPLOR-NIH refinement]]
**[[Rosetta High Resolution Protein Structure Refinement Protocol|ROSETTA refinement]] 

|
*Validation and deposition
**[[PdbStat|PdbStat]]
**[[PSVS|PSVS]]
**[[RPF Analysis|RPF analysis]]
**[[MolProbity Server|MolProbity server]]
**[[RDCvis & KiNG|RDCvis]]
**[[PDB and BMRB Deposition|PDB and BMRB deposition]]
**[[ADIT-NMR|ADIT-NMR]]
**[[HarvestDB|HarvestDB]]
**[[SPINS|SPINS]]

|}

== nmr 2.0 ==

[[http://www.nmr2.buffalo.edu/ Homepage]]

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|

*[http://www.nmr2.buffalo.edu/comm/links/ nmr 2.0 Communication]
*[http://www.nmr2.buffalo.edu/resources/edu/ nmr 2.0 Educational]

*[http://www.nmr2.buffalo.edu/resources/jobs/ nmr 2.0 Jobs]
*[http://www.nmr2.buffalo.edu/resources/jobprofiles/ nmr 2.0 Job Seekers]

*[http://www.nmr2.buffalo.edu/resources/poll/ nmr 2.0 News]

*[http://www.nmr2.buffalo.edu/resources/queries/ nmr 2.0 Queries]

*[http://www.nmr2.buffalo.edu/resources/humor/ nmr 2.0 Humor]

*[http://www.nmr2.buffalo.edu/resources/links/ nmr 2.0 Links]

*[http://www.nmr2.buffalo.edu/blog nmr 2.0 Blog]

|}

For a more linear view of the contents including those in development see [[Wiki Tree Layout]]
*[http://www.nmr2.buffalo.edu/resources/portable/ 18 October protocol]

Routine Processing Procedure for 3D 15N and 13C-edited Experiments

2017-11-16T21:55:19Z

Alex: Reverted edits by Alex (Talk) to last revision by Pwils3

Routine Processing Procedure for 3D 15N and 13C-edited Experiments

2017-11-16T21:55:06Z

Alex: Reverted edits by Pwils3 (Talk) to last revision by Hlee

Aromatic side chain assignment with Aro-HCCH-COSY in XEASY

2017-11-16T21:54:43Z

Alex: Reverted edits by Pwils3 (Talk) to last revision by Jlmills

Aromatic (4,3)D HCCH shows signals for aromatic H-C-C-H moeties. See [[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|aliphatic (4,3)D HCCH Analysis]] for comparison.

Before proceeding, it is helpful to look for the QD (Phe and Tyr), HD2 (His), and HD1 (Trp) resonances in the simultaneous [1H, 1H]-NOESY. Look for a consistent peak that is visible in the amide, alpha, and beta strips near 7ppm.

'''Phe, Tyr, and His'''

*Run the following macro in UBNMR to generate a peaklist and atom list: 
<pre> init
read seq myprot.seq #change filename to something appropriate
read prot noe.prot #change filename to your most current prot list

add GFTatom COSY_C ATTACHED_H

simulate 3D CG COSY_H 0 1
simulate 3D CG COSY_C 0 1
simulate 3D CG1 COSY_CH 0 1
simulate 3D CG1 COSY_C 0 1
simulate 3D CG2 COSY_CH 0 1
simulate 3D CG2 COSY_C 0 1

simulate 3D CD COSY_CH 0 2
simulate 3D CD COSY_C 0 2
simulate 3D CD1 COSY_CH 0 2
simulate 3D CD1 COSY_C 0 2
simulate 3D CD2 COSY_CH 0 2
simulate 3D CD2 COSY_C 0 2

simulate 3D CZ2 COSY_CH 0 3
simulate 3D CZ2 COSY_C 0 3
simulate 3D CZ3 COSY_CH 0 3
simulate 3D CZ3 COSY_C 0 3
simulate 3D CE3 COSY_CH 0 4
simulate 3D CE3 COSY_C 0 4
simulate 3D CH2 COSY_CH 0 4
simulate 3D CH2 COSY_C 0 4

write prot hcchAroI1.prot #the newly created starting prot list
write peaks hcchAroI1.peaks #the newly created starting peak list
</pre>
*In XEASY, use <tt>ns</tt> to load the three sub-spectra of aromatic (4,3)D HCCH; use <tt>ls</tt>, <tt>lp</tt> and <tt>lc</tt> to load the sequence, peak list, and chemical shift list, respectively and <tt>se</tt>, <tt>gs</tt> to sort and display [w1(13C;1H);w3(1H)]-strips.
*In XEASY, use <tt>pm</tt>, <tt>es</tt>, <tt>se</tt> and <tt>gs</tt> (or <tt>sf</tt>) to display [w1(13C;1H);w2(13C)]-planes, and sort and [w1(13C;1H);w2(13CD)]-planes; use <tt>mr</tt> to accurately adjust peak positions to assign 13CD chemical shifts
*use <tt>pm</tt> and <tt>gs</tt> to re-display [w1(13C;1H); w3(1H)]-planes and [w1(13C;1H); w3(1H)]-strips; use <tt>mr</tt> to accurately adjust peak positions to confirm QD chemical shifts. The strips in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, ..., 13CD±QD and 13CD±QE. Use <tt>mr</tt> to accurately position peaks along w1; use <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to save updated PeakList and AtomList.
*In UBNMR, run <tt>updatacosyGFT</tt> to calculate SQ shifts from updated AtomList. Repeat the above steps for the QE<tt>strips</tt> and assign QE. Note that for His, the assignment is often complicated by the presence of strong signals from the His-tag introduced to facilitate protein purification. The QE strip in the basic spectra is expected to exhibit peaks at 13CD, 13Main.CE, 13CZ, ..., 13CD±QD, 13Main.CE±QE and possibly 13CZ±1HZ (for Phe only).

'''TRP'''

*In XEASY, repeat steps 1-3 for strips in the order HH2 > HZ2 > HZ3 > HE3 (instead of the strip order QD > QE for Tyr and Phe). In HH2-strips, assign 13CH2±1HH2, and 13CZ2±1HZ2. In HZ2-strips assign 13CZ2± 1HH2, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3-strips, assign 13CZ3±1HZ3, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3 strip, assign 13CZ3±1HZ3, and 13CE3±1HE3.

CARA vs Xeasy

2017-11-16T21:54:02Z

Alex: Reverted edits by Pwils3 (Talk) to last revision by YunfenHe

Below is concise list of key features of CARA as compared to [[XEASY]] that should help new users make a transition.

==== Data Organization ====

[[XEASY]] keeps data stored in separate files: spectra, sequence lists, atom lists, and peaklists.

CARA stores all data in a "repository" - an XML file, which contains all information (residue type definitions, spectrum types definitions, spin systems, spins, peak lists, extension scripts, paths to NMR spectra, etc.). This file typically has a .cara extension. A repository is built from a template by adding "projects". This approach allows much better control and overview over a structure project and makes sharing data among researchers easier.

Though CARA protects repositories from modifications, which may compromise its stability, repositories can be edited with a plain text or XML editor.

==== Atom Nomenclature ====

Current repository templates are designed to be compatible with the BMRB format. That is, H is used instead of HN, and glycines have HA2/3 instead of HA1/2. This is also the default format of '''cyana.lib '''in [[CYANA]] 2.1.

In addition to that, pseudoatoms are labeled as H* instead of Q* and QQ*. For example, HB is used for QB, HD1/2 for QD1/2, and HD for QQD of Leu. This is compatible with the '''pseudo=2''' setting in [[CYANA]] 2.1

==== Residues and Spin Systems ====

XEASY stores information about spin systems (also referred to as fragments in XEASY manual) and residues in the same *.seq file, and requires somewhat confusing swapping of mapping and fragment numbers for sequence-specific assignment.

CARA clearly distinguishes residues and spin systems as separate classes in repository database. Spin systems can be numbered from 1 upward. They can be linked into fragments by setting successor and predecessor tags. Fragments can be "mapped" to the sequence to see if they match a particular stretch of residues. A spin system becomes assigned when its assignment tag is set, pointing to a certain residue in the sequence.

==== Atoms and Spins ====

What XEASY refers to as atoms are called "spins" in CARA. "Atoms" in CARA form a different data class - it is a member of "residue type" as a part of molecular structure. The database of spins can be exported as an XEASY atom list, and an XEASY atom list can be imported into CARA (provided that the nomenclature matches).

While you may need to use separate atom lists with different spectra and peak lists in XEASY, CARA works with a single set of spins. Spin aliases are used in situations where the same spin has different chemical shifts (see below).

==== Spin Labels ====

Spin labels have the form [?|!]SPINLABEL[+|-x], where SPINLABEL is an alphanumeric string and +x or -x is the "offset". The question mark ? as the first character marks temporary assignments, the exclamation sign "!" - stereospecific assignments. Since + and - characters are reserved to specify offsets, lowercase letters "p" and "m" should be used instead for spin labels in GFT spectra. For example, sequential CA+CB peak should be labeled CApCB-1.

Offset designations in CARA carry more information than in XEASY - they are used to search for sequential neighbors.

==== Peak Inference and Peak Lists ====

Though peak lists in XEASY format can be exported from any spectrum, they only need to be generated for NOESY spectra to provide input to programs like CYANA and AutoStructure. They are not needed for backbone and side-chain assignment.

Peak marks, which you see in spectrum display tools ( "scopes", in this case other than MonoScope ) are displayed at positions, inferred from chemical shifts, and residue type and spectrum type definitions. Spectrum type definitions describe the correlations prouced in a given spectrum and valid spin labels in every dimension. This is equivalent to XEASY peaklists being generated from an atom list on-the-fly. If a peak is moved in CARA, then the chemical shifts of the spins involved are updated instantly. If a new peak is picked, new spins are created. These changes are synchronized across all "scopes".

==== Spectral Folding and Aliasing ====

In CARA all spins and peaks have their exact assignments and positions (in ppm). There are no folding attributes for peaks, as in XEASY. Instead, it is possible to navigate beyond the spectrum edge to display the true unfolded location.

==== Spin Links ====

Spin links do not have a direct equivalent in XEASY. They are designed to describe correlations observed in NOESY spectra, but CARA does not prescribed a unique use for them. Spin links present a good means of visualizing UPLs or short distances in NOESY spectra. Spin links can also be used instead of peak list in manual NOE assignment.

A useful feature is that a single spin link produces two peaks - the direct and transposed. Spin links and inferred peaks can be exported as XEASY peak lists.

==== Spin Aliases ====

There may be significant mismatches between related NMR spectra. Typical causes are different sample and experimental conditions, such as temperature differences or non-resonant effects, or systematic offsets, such as those between TROSY and conventional NMR spectra. In such cases spin aliases can be helpful.

The spin alias object is a child of spin. It has its own chemical shift (typically different from that of spin itself), and a tag with the ID of the spectrum, where this alias was set. Spin aliases have a purely cosmetic effect on spectral appearance, defining positions, where planes and and slices will be taken.

It is recommended to have all spectra calibrated and matched as best as possible, before resorting to spin aliases. Also you would want to have the main chemical shifts set to match the NOESY spectra, since they are exported for use in automated structure calculation, and use aliases for other spectra, such as HCCH-COSY.

==== Scripting ====

CARA functionality is extended by scripts in Lua programming language ([http://www.lua.org/ www.lua.org]). Besides default Lua functionality interface to many CARA functions is available.

-- AlexEletski - 06 Mar 2007

[[Category:CARA]][[Category:Resonance_Assignment]][[Category:XEasy]]

Conditioning procedure for cryogenic probes

2014-02-12T18:18:34Z

Alex: /* Cryogenic Probe Conditioning */

The information below is taken from '''Decoupling Noise''' in [http://www.varianinc.com/image/vimage/docs/products/nmr/apps/pubs/manuals/0199919600c.pdf HCN Cold Probe manual]

== '''Cryogenic Probe Conditioning''' ==

NMR experiments that require X-nucleus decoupling perform best when the rf coils are conditioned. Conditioning imparts just enough energy into the rf coils to disperse any extraneous condensed material into the vacuum space so that the material is carried away by the pump.

Use the following procedures to condition the rf coils if the Cold Probe has been left idle for a number of days or thermal cycled. Both procedures (run in the order presented) are required during the initial installation of the probe.

#Install the probe in the magnet.
#Start cryogenic operations, refer to the Cryogenic Systems Operation and Installation manual.
#Determine the approximate 90º pulse width and powers for all channels. Refer to the contract for pulse widths and use the procedures in Testing Probe NMR Performance, page 19.
#Take the sample out of the probe.
#Proton Channel
##Enter <tt>s2pul temp=25 tn='H1' pw=200 d1=0.1 at=0.1 nt=3000 dp='y'</tt>
##Set <tt>tpwr</tt> to the normal high-power level
##Run experiment with <tt>go</tt>
#X Nucleus Channels - Do this procedure for both X Nucleus Channels.
##Enter <tt>pwxcal</tt>, at the prompts select decoupler channel and nucleus.
##For channel 2 (13C) enter <tt>pw=0 pwx1=200 pwx2=0 temp=25 at=0.1 d1=0.1 nt=3000 dp='y' dm='nnn' dm2='nnn'</tt> For channel 3 (15N) enter <tt>pw=0 pwx1=0 pwx2=200 temp=25 at=0.1 d1=0.1 nt=3000 dp='y' dm='nnn' dm2='nnn'</tt>
##Set <tt>pwxlvl1</tt> or <tt>pwxlvl2</tt> (<tt>dpwr</tt> or <tt>dpwr2</tt>, in older VNMRJ versions) to the corresponding high-power level.
##Run experiment with <tt>go</tt>
#When completed, replace the sample in the magnet. Either monitor the real data as it comes in or set up the experiment but run the first increment a number of times using <tt>array('nt',200,1,0)</tt> to see if the conditioning has been successful.

== '''cryo_noisetest Macro Procedure (Lower Power, Longer Time)''' ==

This is an rf decoupling coil conditioning procedure that also quantifies the noise profile of a Cold Probe. The macro runs forever by cycling between periods of prolonged pulsing and testing.

#Remove the sample from the magnet.
#Ensure that the probe parameter is set to a valid probefile name.
#Enter <tt>cryo_noisetest</tt>. Enter the number of minutes that the decoupler coil conditioning will run before another quantitative test is done. Add five minutes (for the quantitative testing) to the number you entered to get a total recycle time. For example, 40 will run the conditioning for forty minutes followed by five minutes of tests and then start over. Results are printed out at the end of the tests. Reduce the number for more testing (per unit time), increase it for more.
#Enter desired number (use 40 during the initial installation of the probe). The procedure starts and a quantitative test is done straight away as a baseline measurement. Four mini-tests are done consecutively; two carbon and two nitrogen, if more than 2 channels are present. For each nucleus CW and WALTZ decoupling modes are selected. An array of <tt>dpwr</tt> and <tt>dpwr2</tt> is done for each nucleus and for each decoupling mode. The noise is estimated from each spectrum and saved in two date-stamped text files in the local users <tt>vnmrsys/data/testlib</tt> directory and also plotted out in graphical form.
#Enter <tt>aa</tt> or <tt>halt</tt> to stop the acquisition(s) and procedure.

-- Main.AlexEletski - 07 Mar 2008

TALOS

2013-02-03T18:49:59Z

Alex:

== '''Introduction''' ==

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 <ref><pubmed>10212987</pubmed></ref>. In 2009, the Bax laboratory released a new and improved version of the program called TALOS+<ref><pubmed>19548092</pubmed></ref>. 

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.  For installation questions and other support, you can also e-mail [mailto:shenyang@niddk.nih.gov Yang Shen].

 

There is a '''web-based server''' available from Ad Bax's [http://spin.niddk.nih.gov/bax/software/TALOS+/index.html TALOS+] web site: [http://spin.niddk.nih.gov/bax/software/TALOS+/index.html http://spin.niddk.nih.gov/bax/software/TALOS+/index.html] This is probably the best thing to use since you can be sure that the Talos+ chemical shift database is the most recent. 

== '''Generating TALOS dihedral angle constraints with CYANA (UB)''' ==

#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.
#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.
#Start CYANA and type: <pre>read prot XXXX.prot
</pre>

taloslist XXXX

#This will create the TALOS input file <tt>XXXX.tab</tt>. In this file rename all "H" atoms to "HN".
#In a UNIX shell run 
#:talos+ -in XXXX.tab
#:This will create a file <tt>pred.tab</tt>, which includes an initial summary of the prediction results.
#In a UNIX shell run 
#:rama+ -in XXXX.tab
#: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>.
#Start CYANA and type: 
#:talosaco pred #or "talos.tab" -- use the appropriate filename
#:write aco talos.aco
#: 

=== '''talosaco.cya macro''' ===

The <tt>talosaco</tt> macro is invoked as:
<pre>talosaco file [factor [width]]</pre>
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>.

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.

See also the <tt>~/demo/details/TalosAngleRestraints.cya</tt> example script in your local CYANA 2.1 installation.

 

=== '''Interactive Refinement of TALOS Predictions''' ===

Guidelines for refining the TALOS output:

*Classify prediction as <tt>Good</tt> only if
**All 10 best database matches fall in a "consistent" region of the Ramachandran map
**Or 9 out of 10 best database matches fall in a consistent region with <tt>phi < 0</tt>, and the one outlier also lies in <tt>phi < 0</tt> half of the map
**Or 9 out of 10 of the best database matches fall in a consistent region with <tt>phi > 0</tt>
*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).

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.

During structure refinement you can refine TALOS predictions against a preliminary structure.
<pre>vina.tcl -in XXXX.tab -ref XXXX.pdb -auto</pre>
and
<pre>rama.tcl -in XXXX.tab -ref XXXX.pdb</pre>
 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).

 

{| border="1"
|-
| Element
| PHI
| PSI
|-
| α-helix
| -60
| -45
|-
| β-sheet
| -140
| 135
|}

 

=== '''Recommendations for Using TALOS constraints in CYANA calculations''' ===

* The recommended conservative approach is to apply TALOS constraints for regular secondary structure elements (as predicted by CSI, for example) only, excluding the flanking residues.
* If the NOE constraint network is quite dense, the best approach is to run an automated CYANA calculation without TALOS constrains and then verify the TALOS predictions for consistency with the PHI/PSI angle distributions in the resulting structures. The validated TALOS constraints can then be used in subsequent structure calculations.
* TALOS constraints may be used from the beginning to improve convergence of automated CYANA calculations in challenging cases, such as systems with sparse NOE, or homodimeric proteins.

 

== '''Using TALOS and TALOS+ at CABM''' ==

=== Preparing for a TALOS+ run ===

*Make a sub-directory in your project for TALOS.
*you will need the following files in your directory:
*a bmrb file in 2.1 format.  Here is an [[Media:PfR193A_062509_2.1f_4CYANA.bmrb|example]].
*[[Media:BMRBParsing.pm|BMRBParsing.pm]]:  BMRB parser
*[[Media:Tab4Talos.txt|Tab4Talos.pl]]:  perl script to prepare input file for TALOS
*[[Media:Talos2dyana_taloserrors.txt|talos2dyana_taloserrors.pl]]:  perl script to prepare a CYANA .aco file
*Run the following command:
<pre> Tab4Talos.pl [.bmrbf] [input4Talos]

</pre>
This make an input chemical shift list for TALOS.  Here is an [[Media:PfR193A_4Talos.input|example]]. 

=== Running TALOS+ and making a dihedral angle constraint file ===

*Next run talos+:
<pre> talos+ -in [input4Talos]</pre>
This makes a number of output files including the pred.tab.  

*Next, edit the pred.tab and comment out (#) any lines that do not have the "10 Good" comment. 
*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.  They user can modify this script to make his/her own error limits (i.e., +/- 20 or 30).
<pre> perl talos2dyana_taloserrors.pl pred.tab [output.aco]
</pre>
 

== '''References''' ==

<references />

TALOS

2013-02-03T18:36:09Z

Alex:

== '''Introduction''' ==

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 <ref>&amp;amp;amp;amp;amp;lt;pubmed&amp;amp;amp;amp;amp;gt;10212987&amp;amp;amp;amp;amp;lt;/pubmed&amp;amp;amp;amp;amp;gt;</ref>. In 2009, the Bax laboratory released a new and improved version of the program called TALOS+<ref>&amp;amp;amp;amp;amp;lt;pubmed&amp;amp;amp;amp;amp;gt;19548092&amp;amp;amp;amp;amp;lt;/pubmed&amp;amp;amp;amp;amp;gt;</ref>. 

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.  For installation questions and other support, you can also e-mail [mailto:shenyang@niddk.nih.gov Yang Shen].

 

There is a '''web-based server''' available from Ad Bax's [http://spin.niddk.nih.gov/bax/software/TALOS+/index.html TALOS+] web site: [http://spin.niddk.nih.gov/bax/software/TALOS+/index.html http://spin.niddk.nih.gov/bax/software/TALOS+/index.html] This is probably the best thing to use since you can be sure that the Talos+ chemical shift database is the most recent. 

== '''Generating TALOS dihedral angle constraints with CYANA (UB)''' ==

#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.
#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.
#Start CYANA and type: <pre>read prot XXXX.prot
</pre>

taloslist XXXX

#This will create the TALOS input file <tt>XXXX.tab</tt>. In this file rename all "H" atoms to "HN".
#In a UNIX shell run 
#:talos+ -in XXXX.tab
#:This will create a file <tt>pred.tab</tt>, which includes an initial summary of the prediction results.
#In a UNIX shell run 
#:rama+ -in XXXX.tab
#: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>.
#Start CYANA and type: 
#:talosaco pred #or "talos.tab" -- use the appropriate filename
#:write aco talos.aco
#: 

=== '''talosaco.cya macro''' ===

The <tt>talosaco</tt> macro is invoked as:
<pre>talosaco file [factor [width]]</pre>
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>.

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.

See also the <tt>~/demo/details/TalosAngleRestraints.cya</tt> example script in your local CYANA 2.1 installation.

 

=== '''Interactive Refinement of TALOS Predictions''' ===

Guidelines for refining the TALOS output:

*Classify prediction as <tt>Good</tt> only if
**All 10 best database matches fall in a "consistent" region of the Ramachandran map
**Or 9 out of 10 best database matches fall in a consistent region with <tt>phi < 0</tt>, and the one outlier also lies in <tt>phi < 0</tt> half of the map
**Or 9 out of 10 of the best database matches fall in a consistent region with <tt>phi > 0</tt>
*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).

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.

During structure refinement you can refine TALOS predictions against a preliminary structure.
<pre>vina.tcl -in XXXX.tab -ref XXXX.pdb -auto</pre>
and
<pre>rama.tcl -in XXXX.tab -ref XXXX.pdb</pre>
 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).

 

{| border="1"
|-
| Element
| PHI
| PSI
|-
| α-helix
| -60
| -45
|-
| β-sheet
| -140
| 135
|}

 

=== '''Recommendations for Using TALOS constraints in CYANA calculations''' ===

* The recommended conservative approach is to apply TALOS constraints for regular secondary structure elements (as predicted by CSI, for example) only, excluding the flanking residues.
* If the NOE constraint network is quite dense, the best approach is to run an automated CYANA calculation without TALOS constrains and then verify the TALOS predictions for consistency with the PHI/PSI angle distributions in the resulting structures. The validated TALOS constraints can then be used in subsequent structure calculations.
* TALOS constraints may be used from the beginning to improve convergence of automated CYANA calculations in challenging cases, such as systems with sparse NOE, or homodimeric proteins.

 

== '''Using TALOS and TALOS+ at CABM''' ==

=== Preparing for a TALOS+ run ===

*Make a sub-directory in your project for TALOS.
*you will need the following files in your directory:
*a bmrb file in 2.1 format.  Here is an [[Media:PfR193A_062509_2.1f_4CYANA.bmrb|example]].
*[[Media:BMRBParsing.pm|BMRBParsing.pm]]:  BMRB parser
*[[Media:Tab4Talos.txt|Tab4Talos.pl]]:  perl script to prepare input file for TALOS
*[[Media:Talos2dyana_taloserrors.txt|talos2dyana_taloserrors.pl]]:  perl script to prepare a CYANA .aco file
*Run the following command:
<pre> Tab4Talos.pl [.bmrbf] [input4Talos]

</pre>
This make an input chemical shift list for TALOS.  Here is an [[Media:PfR193A_4Talos.input|example]]. 

=== Running TALOS+ and making a dihedral angle constraint file ===

*Next run talos+:
<pre> talos+ -in [input4Talos]</pre>
This makes a number of output files including the pred.tab.  

*Next, edit the pred.tab and comment out (#) any lines that do not have the "10 Good" comment. 
*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.  They user can modify this script to make his/her own error limits (i.e., +/- 20 or 30).
<pre> perl talos2dyana_taloserrors.pl pred.tab [output.aco]
</pre>
 

== '''References''' ==

<references />

Resonance Assignment/CARA

2012-08-08T20:13:21Z

Alex:

The topics below provide an overview of resonance assignment of 15N,13C-labeled proteins using CARA. For more information and tutorials please consult [http://www.nmr.ch CARA web site].

*[[CARA Introduction|Introduction]]
*[[CARA vs Xeasy|Differences from XEASY]]
*[[CARA Scopes|Scopes - Displaying NMR Spectra]]

*[[Resonance Assignment/CARA/Starting a new project|Starting a new project]]
*[[Resonance Assignment/CARA/Backbone assignment|Backbone resonance assignment]]
*Side Chain Assignment
**[[Aliphatic Side Chain Assignment with CARA|Aliphatic side-chain assignment]]
**[[Aromatic Side Chain Assignment with CARA|Aromatic side-chain assignment]]
**[[Amide Side Chain Assignment with CARA|Amide side-chain assignment]]
*[[SSAFromFractional13CLabeledSample|Stereospecific Assignment of Leu and Val Isopropyl Groups]]
*[[Creating NOESY peaklists with CARA|Creating NOESY peaklists]]

Also see [[Resonance_Assignment/CARA/Backbone_assignment_GFT|backbone assignment with GFT spectra]]

Resonance Assignment/CARA

2012-08-08T20:12:57Z

Alex:

The topics below provide an overview of resonance assignment of 15N,13C-labeled proteins using CARA. For more information and tutorials please consult [http://www.nmr.ch CARA web site].

*[[CARA Introduction|Introduction]]
*[[CARA vs Xeasy|Differences from XEASY]]
*[[CARA Scopes|Scopes - Displaying NMR Spectra]]

*[[Resonance Assignment/CARA/Starting a new project|Starting a new project]]
*[[Resonance Assignment/CARA/Backbone assignment|Backbone resonance assignment]]
*Side Chain Assignment
**[[Aliphatic Side Chain Assignment with CARA|Aliphatic side-chain assignment]]
**[[Aromatic Side Chain Assignment with CARA|Aromatic side-chain assignment]]
**[[Amide Side Chain Assignment with CARA|Amide side-chain assignment]]
*[[Creating NOESY peaklists with CARA|Stereospecific Assignment of Leu and Val Isopropyl Groups]]
*[[Creating NOESY peaklists with CARA|Creating NOESY peaklists]]

Also see [[Resonance_Assignment/CARA/Backbone_assignment_GFT|backbone assignment with GFT spectra]]

SSAFromFractional13CLabeledSample

2012-08-08T20:11:48Z

Alex: moved NESG:SSAFromFractional13CLabeledSample to SSAFromFractional13CLabeledSample

== '''Stereospecific assignments with a 13C Fractionally Labeled Sample''' ==

Isopropyl groups of Val and Leu can be sterospecifically assigned with the help of a 5-10% fractionally 13C-labeled sample. In biosynthesis of amino acids from glucose the '''pro-R''' methyl carbon and the quaternary carbon come from one glucose molecule and the '''pro-S''' carbon is taken from another. With low-level fractional 13C-labeleing it is extremely unlikely that both glucose molelules are 13C-labeled. Therefore, '''pro-R''' 13C carbons have a 13C neighbor, but '''pro-S''' do not.

=== '''Determining stereospecific assignments''' ===

Experimental procedure:

# Record constant-time [13C,1H] HSQCs with constant-time delays 28 ms (1/J), 42 ms (1.5/J) and 56 ms (2/J)
# Process all HSQCs with identical phase parameters
# In XEASY or CARA identify '''pro-R''' (QD1/QG1) and '''pro-S''' (*QD2/QG2) groups by observing the following
** QD2/QG2 have the same sign in 28 ms and 56 ms spectra.
** QD1/QG1 change sign in 28 ms and 56 ms spectra.
** Only QD2/QG2 are oberved in 42 ms spectrum.

* 42 ms HSQC is rather insensitive and is optional. It is useful under conditions of severe overlap of methyl groups, when peaks of opposite sign tend to cancel each other in the 28 ms HSQC.
* 56 ms could also be skipped if the target protein has assigned Met methyl groups. Since they do not have any directly bonded carbon spins, their peaks have the same sign in the 28 ms HSQC as the '''pro-S''' (QD2/QG2) groups.

Reference:

[http://pubs.acs.org/cgi-bin/archive.cgi/bichaw/1989/28/i19/pdf/bi00445a003.pdf Neri et al. Biochemistry 28 (19): 7510-7516 SEP 19 1989]

=== '''Applying stereospecific assignments of methyl groups''' ===

It is recommended to use stereospecific assignments of methyl groups before running the FOUND module or prior to doing automated structure calculation. This improves accuracy and precision of the initial structure calculation.

It is also possible to apply them later at the refinement stage prior to using GLOMSA if spectra of the fractionally labeled sample were not recorded in time for automated structure calculation.

You can create CYANA scripts as described in the topic about [[NESG:ApplyingSSA|applying stereospecific assignments]]. Use a swapped atom list and a separate <tt>stereo.cya</tt> file. For better transparency in a given project it is better to swap assignments in the original file (XEASY atom list or CARA repository), rather than relying on swapping them in CYANA.

To swap methyl spin labels in CARA do the following for Leu (or Val):
# In the [13C,1H]-HSQC plane in PolyScope select HD1/CD1 (or HG1/CG1) spins from the list.
# Right-click and select '''Label Spins'''
# In the pup-up '''Edit Spin Label''' window change the labels to HD/CD (or HG/CG).
# Repeat steps 1-3 to change HD2/CD2 to HD1/CD1 (or HG2/CG2 to HG1/CG1).
# Repeat steps 1-3 to change HD/CD to HD2/CD2 (or HG/CG to HG2/CG2).

To swap methyl spin labels in XEASY do the following for Leu (or Val):
# Open the current .prot list in a text editor
# Change the atom numbers of the QD1/QD2 '''and''' CD1/CD2 (or QG1/QG2 and CG1/CG2)
<nowiki>
646 0.831 0.000 QD1 39 #swap 646 and 647
647 0.724 0.000 QD2 39 #swap 646 and 647
648 25.020 0.000 CD1 39 #swap 648 and 652
649 999.000 0.000 HD11 39
650 999.000 0.000 HD12 39
651 999.000 0.000 HD13 39
652 22.667 0.000 CD2 39 #swap 648 and 652
</nowiki>
# Reload the .prot list in all open spectra

%COMMENT%

-- Main.AlexEletski - 13 Jun 2007

Conditioning procedure for cryogenic probes

2012-05-09T17:55:47Z

Alex: /* Cryogenic Probe Conditioning */

The information below is taken from '''Decoupling Noise''' in [http://www.varianinc.com/image/vimage/docs/products/nmr/apps/pubs/manuals/0199919600c.pdf HCN Cold Probe manual]

== '''Cryogenic Probe Conditioning''' ==

NMR experiments that require X-nucleus decoupling perform best when the rf coils are conditioned. Conditioning imparts just enough energy into the rf coils to disperse any extraneous condensed material into the vacuum space so that the material is carried away by the pump.

Use the following procedures to condition the rf coils if the Cold Probe has been left idle for a number of days or thermal cycled. Both procedures (run in the order presented) are required during the initial installation of the probe.

#Install the probe in the magnet.
#Start cryogenic operations, refer to the Cryogenic Systems Operation and Installation manual.
#Determine the approximate 90º pulse width and powers for all channels. Refer to the contract for pulse widths and use the procedures in Testing Probe NMR Performance, page 19.
#Take the sample out of the probe.
#Proton Channel
##Enter <tt>s2pul temp=25 tn='H1' pw=200 d1=0.1 at=0.1 nt=3000 dp='y'</tt>
##Set <tt>tpwr</tt> to the normal high-power level
##Run experiment with <tt>go</tt>
#X Nucleus Channels - Do this procedure for both X Nucleus Channels.
##Enter <tt>pwxcal</tt>, at the prompts select decoupler channel and nucleus.
##For channel 2 (13C) enter <tt>pw=0 pwx1=200 pwx2=0 temp=25 at=0.1 d1=0.1 nt=3000 dp='y' dm='nnn' dm2='nnn'</tt> For channel 3 (15N) enter <tt>pw=0 pwx1=0 pwx2=200 temp=25 at=0.1 d1=0.1 nt=3000 dp='y' dm='nnn' dm2='nnn'</tt>
##Set <tt>dpwr</tt> or <tt>dpwr2</tt> to the corresponding high-power level.
##Run experiment with <tt>go</tt>
#When completed, replace the sample in the magnet. Either monitor the real data as it comes in or set up the experiment but run the first increment a number of times using <tt>array('nt',200,1,0)</tt> to see if the conditioning has been successful.

== '''cryo_noisetest Macro Procedure (Lower Power, Longer Time)''' ==

This is an rf decoupling coil conditioning procedure that also quantifies the noise profile of a Cold Probe. The macro runs forever by cycling between periods of prolonged pulsing and testing.

#Remove the sample from the magnet.
#Ensure that the probe parameter is set to a valid probefile name.
#Enter <tt>cryo_noisetest</tt>. Enter the number of minutes that the decoupler coil conditioning will run before another quantitative test is done. Add five minutes (for the quantitative testing) to the number you entered to get a total recycle time. For example, 40 will run the conditioning for forty minutes followed by five minutes of tests and then start over. Results are printed out at the end of the tests. Reduce the number for more testing (per unit time), increase it for more.
#Enter desired number (use 40 during the initial installation of the probe). The procedure starts and a quantitative test is done straight away as a baseline measurement. Four mini-tests are done consecutively; two carbon and two nitrogen, if more than 2 channels are present. For each nucleus CW and WALTZ decoupling modes are selected. An array of <tt>dpwr</tt> and <tt>dpwr2</tt> is done for each nucleus and for each decoupling mode. The noise is estimated from each spectrum and saved in two date-stamped text files in the local users <tt>vnmrsys/data/testlib</tt> directory and also plotted out in graphical form.
#Enter <tt>aa</tt> or <tt>halt</tt> to stop the acquisition(s) and procedure.

-- Main.AlexEletski - 07 Mar 2008

Refinement Strategies

2012-05-01T02:59:47Z

Alex:

Let's assume that at this stage you have
* Nearly complete resonance assignments
* Unassigned NOESY peaklist (possibly automatically picked)
* Converged automatically calculated structure (for example, using CYANA)

Subsequent structure refinement involves:
* Resonance assignment verification and completion
** Search for missing assignments based on initial structure (predicted short distance) and NOESY peaks that could not be assigned during automated structure calculation
** Slowly exchanging hydroxyl protons of Ser, Thr and Tyr, as well as thiol protons of Cys can be identified in the same manner.
* Peak list optimization
** Pick missed NOE peaks and adjust peak positions for existing peaks based on the initial structure. This is especially important for automatically generated peaklists. In CARA, the convenient way is to load short distances as 'SpinLinks' and display them along with peaklists for guidance.
** Remove any peaks caused by spectral artifacts, such as noise ridges, axial peaks, solvent lines, truncation wiggles, etc.
** Search output peaklists (such as '*-cycle7.peaks' from CYANA) for strong unassigned NOE peaks. Unless caused by obvious spectral artifacts, these most likely indicate incorrect or missing resonance assignments. Other causing factors could be convergence to an incorrect initial structure (more common for dimers), and exchange peaks if multiple conformations are present.
** Revise peak integration and/or calibration constants, if necessary.
* TALOS verification
** When using TALOS dihedral angle constraints verify TALOS prediction against the initial structure using the Rama viewer.
* Refinement using orientational constraints from RDCs

Optimized resonance assignments and NOE peaklists can then be used in additional rounds of automated structure calculation.

Recommendations for refinement of NOE peak assignments
* 'Consensus' peaklists - verify cases of assignment disagreement
* Temporarily tighten NOE calibration and run calculation with fixed unambiguous assignments (option 'KEEP' in noeassign). The strongest NOE violations would correspond to peaks with assignment issues. If violations are reported only for otherwise legitimate peaks, the calibration constants are too tight.

Refinement Strategies

2011-10-25T16:26:31Z

Alex: Created page with '* Peak list optimization and integration * Resonance assignment verification and completion'

* Peak list optimization and integration
* Resonance assignment verification and completion

Main Page

2011-10-25T16:23:49Z

Alex: /* Structure Calculation and Validation */

__NOTOC__

<big>'''Welcome to the NESG Wiki!'''</big> 

The NESG Wiki is a medium for sharing experimental protocols as well as training an educational materials in the fields of structural biology, structural genomics and biomolecular NMR.

Please check out [[NESG NMR wiki workshop at the 2010 Keystone meeting|NESG NMR wiki workshop presentations at the 2010 Keystone meeting]]

== Protein Sample Production ==

{| cellspacing="1" class="FCK__ShowTableBorders"
|- valign="top"
|
*[[Target selection|NESG target selection]] 
*[[DNA cloning protocols|DNA cloning protocols]] 
*[[Protein purification|Protein expression and purification protocols]]  
*Sample Optimization
**[[Construct optimization]]
**[[Buffer optimization]]
**[[Cofactor optimization]]

|
*Protein Sample Analysis
**[[SDS page gel]]
**[[Protein concentration|Protein concentration measurements]]
**[[Oligomerization Status|Assessment of Oligomerization State]]
***[[Gel filtration and light scattering|gel-filtration and light scattering]]
***[[NMR determined Rotational correlation time]]
**[[MassSpectrometry|Mass spectrum]]
**[[NMR screening]]

 

|}

== NMR Data Acquisition ==

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
*Routine operation
**[[NMR Sample Preparation]]
**[[Inserting NMR Sample]]
**[[Tuning and matching]]
**[[Deuterium Lock]]
**[[Shimming]]
**[[Pulse width calibration]]
**[[Temperature calibration]]
**[[Chemical shift referencing]]
*Advanced operation
**[[Deuterium pulse width calibration and decoupling]]

|
*NMR data acquisition for protein structure determination
**[[Common NMR experiment sets]]
**[[NMR experiment setup scripts for VNMRJ|Custom NMR experiment setup scripts for VNMRJ]]
**[[Estimation of rotational correlation time]]
**[[Estimation of measurement time]]
**[[Simultaneous 13C,15N-resolved NOESY]]
**[[2D (13C, 1H) HSQC for fractionally 13C-labeled samples|2D [13C, 1H]-HSQC for fractionally 13C-labeled samples]]
**[[Long-range 15N-1H correlation experiments for histidine rings]]
*[[Setting up non-uniformly sampled spectra|Non-uniform sampling (NUS) ]]
**[[Setting up non-uniformly sampled spectra/NUS guide for Varian|NUS - Varian]]
**[[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|NUS - Bruker]]

|
*Maintenance (VARIAN)
**[[Installing and updating BioPack]]
**[[Full probefile calibration]]
**[[Rebooting spectrometer console]]
**[[Conditioning procedure for cryogenic probes]]

|}

== NMR Data Processing ==

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
NMRPipe

*[[Brief description of philosophy, commands, and functions of NMRPipe|Brief description of philosophy, commands, and functions]]
*[[Routine 2D Experiment|2D experiments]]
*[[Routine Processing Procedure for 3D 15N and 13C-edited Experiments|3D 15N and 13C-edited experiments]]
*[[HSQCTROSY RDC Measurement|2D ]][[HSQCTROSY RDC Measurement|15]][[HSQCTROSY RDC Measurement|N HSQC-TROSY experiment for RDC measurement]]
*[[Jmodulation Experiment RDC|2D J-modulation experiment for RDC measurement]]
*[[Alignment Media Preparation|Alignment Media Preparation for RDC measurement]]

|
Other

*[[Processing NMR spectra with PROSA|PROSA]]
*[[Bruker Data Processing|TOPSPIN]]
*[[AGNuS/AutoProc|AUTOPROC]]
*[[Processing non-uniformly sampled spectra with Multidimensional Decomposition|Processing NUS spectra with MDD]]
*[[Spectra Format Conversion from NMRPipe Data|NMRPipe processed data conversion to Sparky, CARA, XEASY, and NMRViewJ]]

|}

== NMR Resonance Assignment ==

*[[Resonance Assignment/Principles and concepts|Principles and concepts]]

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
*Semi-automated protocols
**[[Resonance Assignment/CARA|CARA]]
**[[Sparky]]
**[[Resonance Assignment/XEASY|XEASY]]

|
*Automated resonance assignment
**[[AutoAssign|AutoAssign]]
**[[AutoAssign WebServer|AutoAssign server]]
**[[Abacus|ABACUS]]
**[[The PINE Server|PINE server]]

|
*Validation and deposition
**[[AVS|Assignment validation suite (AVS)]]
**[[LACS|Linear analysis of chemical shift (LACS)]]
**[[PDB and BMRB Deposition#Preparing_files_for_BMRB_depostion|Depositing chemical shifts]]

|}

== Structure Calculation and Validation ==

[[Structure Calculation and Validation|Principles and concepts]]

{| class="FCK__ShowTableBorders" cellspacing="1"
|- valign="top"
|
*Structure calculation
**[[CYANA Structure Determination Program|CYANA]]
**[[AutoStructure Structure Determination Program|AutoStructure]]
**[[Structure Calculation Using CS-Rosetta|CS-ROSETTA]]
**[[Structure Calculation Using CS-DP ROSETTA|CS-DP ROSETTA]]
**[[Structure Calculation Using CS-RDC-ROSETTA|CS-RDC-ROSETTA]]
**[[Consensus Approaches|"Consensus" approaches]]
**[[Refinement Strategies|Refinement Strategies]]

|
*Special topics
**[[Protein-Ligand Complexes|Protein-Ligand complexes]]
**[[Working With Metal Ions|Metal ions]]
**[[Residual Dipolar Couplings in Structure Refinement|Residual Dipolar Couplings]]
**[[REDCAT|REDCAT]] and [[REDCRAFT|REDCRAFT]]
**[[Paramagnetic Constraints in Structure Determination|Paramagnetic constraints]]
**[[RDC-Assisted Dimer Structure Determination|RDC-assisted dimer structure calculation]]

|
*Structure Refinement
**[[Structure Refinement Using CNS Energy Minimization With Explicit Water|CNS refinement]]
**[[Structure Refinement Using XPLOR-NIH|XPLOR-NIH refinement]]
**[[Rosetta High Resolution Protein Structure Refinement Protocol|ROSETTA refinement]]

|
*Validation and deposition
**[[PdbStat|PdbStat]]
**[[PSVS|PSVS]]
**[[RPF Analysis|RPF analysis]]
**[[MolProbity Server|MolProbity server]]
**[[PDB and BMRB Deposition|PDB and BMRB deposition]]
**[[ADIT-NMR|ADIT-NMR]]
**[[HarvestDB|HarvestDB]]
**[[SPINS|SPINS]]

|}

For a more linear view of the contents including those in development see [[Wiki Tree Layout]]

CYANA

2011-10-25T15:23:34Z

Alex: /* Residue Library */

== '''Introduction''' ==

CYANA is a macromolecular structure calculation algorithm based on simulated annealing molecular dynamics calculations in torsional angle space, in contrast to Cartesian space [1,2].  Here the only degrees of freedom are torsion angles with covalent structure parameters kept fixed, thereby significantly decreasing the number of degrees of freedom in the calculation.

The lateest version of CYANA is 3.0, which is capable of handling orientational (i.e., RDC) constraints. You can find reference material, examples and tutorial at the [http://www.cyana.org/wiki/index.php/CYANA_3.0_Reference_Manual CYANA 3.0 wiki].

Below we provide a more detailed description of input files and protocols as used in the NESG.

 

== '''CYANA 2.1''' ==

=== Residue Library ===

CYANA versions 2.0 and later use a new residue library <tt>~/lib/cyana.lib</tt>. The description of its file format can be found here: [http://www.cyana.org/wiki/index.php/Residue_library_file Residue_library_file] It is loaded by the <tt>cyanalib</tt> command. For backwards compatibility the old DYANA residue library <tt>dyana.lib</tt> is provided (loaded with <tt>dyanalib</tt>, of course).

The main difference is larger van der Waals radii in the newer library. This will give you a larger target function than DYANA, but also better clash scores.

The new library no longer includes separate entries for neutral and charged arginine, lysine, histidine, aspartic and glutamic acid. The new ARG, LYS, ASP and GLU entries in <tt>cyana.lib</tt> correspond to ARG+, LYS+, ASP- and GLU- entries in <tt>dyana.lib</tt>. The HIS residue in <tt>cyana.lib</tt> is a delta-protonated neutral species. The charged (HIS+) and epsilon-protonated neutral (HIST) residues are included in the <tt>special.lib</tt> residue library.

=== Atom Nomenclature ===

Atom nomenclature was made compatible with BMRB standard. The deviations from XEASY/DYANA conventions are: HN <-> H, HA1 <-> HA2 and HA2 <-> HA3 for GLY.

There a is macro '''translate.cya''', which is used to convert input to different formats. For example, to read files with DYANA nomenclature, enter <tt>translate dyana</tt>. To switch back to CYANA 2.1 convention type <tt>translate off</tt>.

 

=== Pseudoatom Treatment ===

Pseudoatom handling is switched by setting '''pseudo=x''', where x is 0, 1, 2, or 3.

With '''pseudo=0''', the default setting, coordinate files *.cor and *.pdb do not contain pseudoatoms. They are calculated implicitly on the run.

Setting '''pseudo=1''' restores the old DYANA behavior with explicit pseudoatoms.

Setting '''pseudo=2''' switches to simplified pseudoatom names, such as HB instead of QB, HD1 instead of QD1, and HD instead of QQD of Leu. This is the setting to be used when reading chemical shifts from CARA. Coordinate files will contain explicit pseudoatoms, as with '''pseudo=1'''

Setting '''pseudo=3''' allows X-Plor/CNS pseudoatom names, like HX* instead of QX. For some reason using '''translate xplor''' is not enough to do the conversion for all the atoms.

 

=== The Initialization File:  init.cya ===

The init.cya is a local initialization file, which is read when cyana starts. It should be located in the directory where CYANA is run. In a given project the same file can be used for nearly all calculations.

Create your own init.cya file with the following lines in a text editor or download this template [[Media:CYANA_init.cya|init.cya]] file: 
<pre>name:=XXXX # Replace XXXX with NESG ID
nproc=2 # Number of processors on a workstation
rmsdrange:=20..72 # RMSD reported for these residues after structure calculation
# Read the standard and special libraries
cyanalib
read lib $cyanadir/lib/special.lib append
pseudo=2 # Allows HB, HD, etc. pseudoatom names, use with CARA
read seq $name # Initialize
</pre>
Replace XXXX with your NESG target ID. It is convenient to have the sequence and atomlist files named as <tt>XXXX.seq</tt> and <tt>XXXX.prot</tt>.

*<tt>nproc</tt> defines the number of processors on a workstation.
*<tt>rmsdrange</tt> is only used in structure calculation. Set the range to a valid residue range. From NOE patterns you can exclude flexible N- and C-terminal parts. If you have a flexible loop in the middle you can specify the range as <tt>10..30,40..70</tt>.
*<tt>cyanalib</tt> reads the default <tt>cyana.lib</tt> residue library. The <tt>special.lib</tt> library is appended to use non-standard residues, (i.e., His tautomers).
*<tt>pseudo=2</tt> is only necessary to read atom lists created with CARA, because they have H* for pseudoatom labels. Comment it out, or use <tt>pseudo=0</tt> if you take atom lists from XEASY. 

 

=== The Structure Calculation File:  CALC.cya ===

There are calculation demos for automatic assignment (~/demo/auto) and simple structure calculation (~/demo/manual) runs.

Here is a CALC.cya script for automatic NOE assignment: 
<pre>peaks  := c13.peaks,n15.peaks,aro.peaks # names of NOESY peak lists
prot  := demo # names of chemical shift lists
constraints := demo.aco # additional (non-NOE) constraints
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..100 # residue range for RMSD calculation
randomseed  := 434726 # random number generator seed

noeassign peaks=$peaks prot=$prot autoaco
</pre>
To prevent CYANA from changing existing peak assignments you need to define a subroutine to select the peaks to keep: 
<pre>peaks  := c13.peaks,n15.peaks,aro.peaks # names of NOESY peak lists
prot  := demo # names of chemical shift lists
constraints := demo.aco # additional (non-NOE) constraints
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..100 # residue range for RMSD calculation
randomseed  := 434726 # random number generator seed

subroutine KEEP
peaks select "*, * number=2..7999"
end

noeassign peaks=$peaks prot=$prot autoaco keep=KEEP
</pre>
Here, subroutine <tt>KEEP</tt> is used to keep the assignments for peaks with peak numbers from 2 to 7999.

 

Here is a CALC.cya script for manual structure calculation: 
<pre>peaks  := c13,n15,aro # names of peak lists
prot  := demo # names of proton lists
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)
calibration:= 6.7E5,8.2E5,8.0E4 # calibration constants (will be determined
# automatically, if commented out)
dref  := 4.2 # average upper distance limit for
# automatic calibration

if (master) then

# ---- check consistency of peak and chemical shift lists----

peakcheck peaks=$peaks prot=$prot

# ---- calibration ----

calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref
peaks calibrate "**" simple
write upl $name-in.upl
distance modify
write upl $name.upl

end if
synchronize

# ---- structure calculation ----

read seq $name.seq # re-read sequence to initialize
read upl $name.upl # read upper distance limits
read aco $name.aco # read angle constraints
seed=5671 # random number generator seed
calc_all structures=100 command=anneal steps=10000 # calculate 100 conformers
overview $name.ovw structures=20 pdb # write overview file and coordinates
</pre>
Note the order in which tolerances are given.

The '''calibration''' field can be left empty, in this case '''dref''' will be used to derive calibration constants. If '''dref''' is not specified noeassign.cya will use a default value of 4.0. During calculation noeassign.cya will also relax the calibration if needed (that is in "elastic" mode, which is the default).

'''constraints''' need not be non-NOE despite what the comment says. You can add *.aco, *.upl, *.lol, and even *.cya macros for stereospecific assignments (haven't tested it yet, but that's the way CYANA adds stereospecific assignments in the final round).

'''master''' and '''synchronize''' keywords are needed for running on a cluster.

'''peakcheck''' checks the peaklist assignments against the atom list. Always check CYANA output for '''peakcheck''' results - those huge upl violations may be caused by mis-assigned peaks.

 

=== NOE Calibration ===

CYANA 2.1 by default does not use explicit pseudoatom corrections in distance constraints. Instead, these corrections are applied implicitly on-the-fly. This behavior is turned on by setting '''expand=1'''.

Calibration is thus performed with the undocumented statement '''peaks calibrate "**" simple'''. Trivial calculations show, however, that this command uses a simple r^-6 calibration without adding pseudoatom corrections.

Old calibration macros, such as '''calibrate.cya''' and '''caliba.cya''' are still allowed, but they do add explicit pseudoatom corrections. So if want to use them, don't forget to set '''expand=0'''. Omitting it will result in applying corrections twice, making the corresponding constraints very loose.

This is, of course, a matter of huge confusion since both methods produce otherwise identical *.upl files. Be sure you know HOW you calibrate your NOEs.

To modify upper and lower distance cutoffs for NOE calibration, use '''set upl_values:=2.4,6.0'''. The defaults are 2.4 and 5.5.

For a more detailed description of NOE calibration using CYANA, follow this [[NOE Calibration Using CYANA|link]].

 

=== Dihedral Angle Constraints ===

In CYANA, dihedral angle constraints are specified in a <tt>.aco</tt> file.

Dihedral angle constraints for structure calculation in CYANA can come from a variety of sources.  For example, the [[FOUND|FOUND]] module derives dihedral angle constraints based on local NOE data.

Programs such as [[TALOS|TALOS]] provide backbone phi and psi torsion angle constraints based on chemical shifts.  In our structure determination pipeline we often make use of TALOS-derived backbone torsion angle constraints in our calculations. 

 

=== Stereospecific Assignments ===

Constraints for diastereotopic atoms (such as HB2, HB3) are treated as ambiguous by CYANA. This is switched on with '''swap=1'''.

For the manual run you may want to have '''swap=0''' to be compatible with DYANA behavior. This option is apparently not necessary when distance modification is applied.

Distance modification does not affect Phe and Tyr ring atoms HD1/2 and HE1/2. Therefore, if you have degenerate ring chemical shift (as is almost always the case) make sure you have them labeled QD and QE

External stereospecific assignments determined with [[GLOMSA|'''GLOMSA''']] or with the help of a fractionally (i.e., 5%) 13C-labeled sample [3] can be defined with a custom macro like this: 
<pre> # VAL
atom stereo "QG1 25 36 38 87"
atom stereo "QG1 43 90"
atom swap "QG1 43 90"
# LEU
atom stereo "QD1 60 63 97"
atom stereo "QD1 35 56"
atom swap "QD1 35 56"
</pre>
Here the syntax of CYANA 2.1 requires double quotes. For some strange reason, methyl groups should be written with the letter "Q" even if '''pseudo=2''' is used.

 

=== NOESY Peak Lists ===

CYANA 2.1 can produce multiple assignments for a peak. Below is a part of an aliphatic NOESY peaklist with peak #6 having two assignments. #VC tags specify the weights given to individual assignments. Calibration of this peak yields two constraints splitting the peak integral according to these weights. 
<pre># Number of dimensions 3
#FORMAT xeasy3D
#INAME 1 H
#INAME 2 C
#INAME 3 h
#CYANAFORMAT HCh
1 4.147 51.731 1.474 3 U 7.953E+03 0.000E+00 - 0 2234 2233 2238 #QU 1.000 #SUP 1.00
2 4.147 51.731 4.251 4 U 4.181E+03 0.000E+00 - 0 0 0 0
3 4.147 51.731 7.791 3 U 6.017E+03 0.000E+00 - 0 2234 2233 232 #QU 1.000 #SUP 1.00
4 1.474 22.186 0.515 3 U 1.481E+03 0.000E+00 - 0 2390 2389 1417 #QU 0.981 #SUP 0.98
5 1.474 22.186 1.249 3 U 2.610E+04 0.000E+00 - 0 2390 2389 1706 #QU 0.987 #SUP 0.99
6 1.474 22.186 2.635 3 U 1.396E+03 0.000E+00 - 0 2390 2389 1715 #VC 0.47897 #QU 0.774 #SUP 0.96
2390 2389 1815 #VC 0.52103 #QU 0.813 #SUP 0.96
7 1.474 22.186 3.863 3 U 1.418E+04 0.000E+00 - 0 2390 2389 1657 #QU 0.885 #SUP 0.88
8 1.474 22.186 4.147 3 U 1.448E+04 0.000E+00 - 0 2238 2237 2234 #QU 1.000 #SUP 1.00
</pre>
The peaklists produced by CYANA 2.1 are not backwards-compatible with XEASY, but there are Lua scripts, which can read them into CARA including the information on ambiguous assignments. UBNMR should also be able to handle them in the future.

When supplying completely unassigned peaks for automatic NOE assignment it is necessary to include a line like '''#CYANAFORMAT HCh''' in the header.

 

== '''CYANA 3.0''' ==

Again please consult the [http://www.cyana.org/wiki/index.php/CYANA_3.0_Reference_Manual CYANA 3.0 wiki] for complete details on file formats, input files for CYANA, and other documentation.

=== Residue Library ===

A [http://www.cyana.org/wiki/index.php/Residue_library_file residue library] defines all properties of a residue including atom types, the nomenclature, the dihedral angle definitions, the covalent connectivities and the standard geometry. The standard geometry of the ECEPP/2 force field [4,5] is used for all amino acid residue types.  Standard residues are collected in the '''cyana.lib''' library; special residue types are in the '''special.lib''' library.

=== Sequence File ===

The [http://www.cyana.org/wiki/index.php/Sequence_file sequence file] (.seq) defines the sequence of the molecule you are working with.  Special residue types (i.e., oxdized cysteine, histidine tautomers, and cis-peptide bonds) can also be defined in the sequence file as follows:

*oxidized cysteine:  CISS
*charged histidine:  HIS+
*Nε2H neutral tautomer:  HIST (the default HIS specifies the Nδ1H neutral tautomer).
*cis-peptide bond:  place a "c" before the residue name;  i.e., cPRO
*invisible intermolecular linkers:  PL, LL, LL2, LL5, LP

 

=== Automated Structure calculation ===

<pre>
peaks := n,ali,aro # names of NOESY peak lists
prot := $name # names of chemical shift lists
restraints := talos.aco,stereo.cya # additional (non-NOE) constraints
tolerance := 0.04,0.02,0.4 # chemical shift tolerances
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)
upl_values := 2.4,5.5 # calibration cutoffs
cut_upl=0.05
calibration_constant:= # NOE calibration parameters
structures := 100,20 # number of initial, final structures
steps := 10000 # number of torsion angle dynamics steps
rmsdrange := 20..102 # residue range for RMSD calculation
randomseed := 562 # random number generator seed
calibration_dref := 4.0 # average distance for calibration, default 4.0
keep := # set to KEEP to retain existing assignments

weight_rdc = 0.002 # weight for RDC restraints
cut_rdc = 1.0 # cut-off for RDC violations
opt_tensor = 1 # alignment tensor optimization

subroutine KEEP
peaks select "*,*"
end

noeassign peaks=$peaks prot=$prot keep=$keep autoaco
</pre>

=== '''A Simple Automated Structure Calculation Using CYANA 3.0''' ===

This section provides an example of a standard automatic NOESY assignment calculation using CYANA 3.0 on a monomeric protein.

==== Input Files ====

Collect the following files in your directory (see the attached files for examples and formatting): 

*[[Media:CYANA30ex_init.cya|init.cya]]:  initialization file. Defines the protein name (i.e., PROT), residue library(ies), number of processors used, and rmsd residue range.
*[[Media:CYANA3ex_CALC.cya|CALC.cya]]:  structure calculation file: Defines the peak lists, tolerances, any NOE calibration parameters (default is automatic calibration), total number of structures calculated in each cycle, number of structures with lowest target function retained after each cycle, number of torsion angle dynamics steps, random seed.
*[[Media:CYANA3ex_PfR193A.seq|PROT.seq]]:  protein sequence file.
*[[Media:CYANA3ex_PfR193A.aco|PROT.aco]]:  dihedral angle constraint file.
*[[Media:CYANA3ex_PfR193A_h2o.prot|filename.prot]]:  chemical shift assignment list.  You should make all degenerate geminal proton assignments Q's, as well as degenerate side chain aromatics (HD1/HD2 and HE1/HE2).
**if your assignments are in a bmrb file (2.1), start cyana, read in the bmrb file, and then write the shifts out to a prot file as follows:
<pre> Open project in cyana 3.0:
read bmrb [finename.bmrb]
write prot [filename.prot]</pre>
*[[Media:CYANA3ex_PfR193A_n15.peaks|filename1.peaks]], [[Media:CYANA3ex_PfR193A_c13_h2o_ali.peaks|filename2.peaks]], [[Media:CYANA3ex_PfR193A_c13_h2o_aro.peaks|filename3.peaks]]:  NOESY peak lists in XEASY format.  The peak lists are unassigned.
*[[Media:CYANA3ex_ssa.cya|ssa.cya]]:  file with stereospecific assignments defined.
*other:  other constraint files such as, manual upper and lower distance limits, hydrogen bond constraints.

==== Running the Program ====

To run CYANA 3.0 on our cluster at CABM, login to master3 and type:
<pre> /farm/software/cyana3.0/bin/cyana CALC > log.out
</pre>
==== Output Files ====

A CYANA structure calculation run will produce .pdb, .upl, .noa and .ovw files for each cycle and the final cycle, as well as log and ramachandran files for the run.

The command <tt>cyanatable</tt> produces a summary table of an automated NOE assignment structure calculation run.

 

== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/9367762?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2 1.    Güntert, P,, Mumenthaler, C. and Wüthrich, K. (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. ''J. Mol. Biol. 273'', 283-298.]

[http://www.ncbi.nlm.nih.gov/pubmed/12051947?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=5 2.    Herrmann, T., Güntert P. and Wüthrich, K. (2002) Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA.  ''J. Mol. Biol. 319'' , 209-227.] 

[http://www.ncbi.nlm.nih.gov/pubmed/2692701?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=9 3.    Neri, D., Szyperski, T., Otting, G., Senn, H. and Wüthrich, K. (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. ''Biochemistry 28'', 7510-7516.]

4.    Momany, F.A., McGuire, R.F., Burgess, A.W. and Scheraga, H.A. (1975)  Energy parameters in polypeptides. VII. Geometric parameters, partial atomic charges, nonbonded interactions, hydrogen bond interactions, and intrinsic torsional potentials for the naturally occurring amino acids.  ''J. Phys. Chem. 79'', 2361-2381.

5.    Nemethy, G., Pottle, M.S. and Scheraga, H.A. (1983)  Energy parameters in polypeptides. 9. Updating of geometrical parameters, nonbonded interactions, and hydrogen bond interactions for the naturally occurring amino acids.  ''J. Phys. Chem. 87'', 1883-1887.

CYANA

2011-10-25T15:07:19Z

Alex: /* Introduction */

== '''Introduction''' ==

CYANA is a macromolecular structure calculation algorithm based on simulated annealing molecular dynamics calculations in torsional angle space, in contrast to Cartesian space [1,2].  Here the only degrees of freedom are torsion angles with covalent structure parameters kept fixed, thereby significantly decreasing the number of degrees of freedom in the calculation.

The lateest version of CYANA is 3.0, which is capable of handling orientational (i.e., RDC) constraints. You can find reference material, examples and tutorial at the [http://www.cyana.org/wiki/index.php/CYANA_3.0_Reference_Manual CYANA 3.0 wiki].

Below we provide a more detailed description of input files and protocols as used in the NESG.

 

== '''CYANA 2.1''' ==

=== Residue Library ===

CYANA 2.1 uses a new library ~/lib/cyana.lib. It is loaded by the <tt>cyanalib</tt> command. For back-compatibility there is also the old DYANA library dyana.lib (loaded with <tt>dyanalib</tt>, of course).

The main difference is larger van der Waals radii. This will give you a larger target function than DYANA, but also better clash scores.

Residue nomenclature is also different - there are no charged species, like ARG+ or GLU-.

 

=== Atom Nomenclature ===

Atom nomenclature was made compatible with BMRB standard. The deviations from XEASY/DYANA conventions are: HN <-> H, HA1 <-> HA2 and HA2 <-> HA3 for GLY.

There a is macro '''translate.cya''', which is used to convert input to different formats. For example, to read files with DYANA nomenclature, enter <tt>translate dyana</tt>. To switch back to CYANA 2.1 convention type <tt>translate off</tt>.

 

=== Pseudoatom Treatment ===

Pseudoatom handling is switched by setting '''pseudo=x''', where x is 0, 1, 2, or 3.

With '''pseudo=0''', the default setting, coordinate files *.cor and *.pdb do not contain pseudoatoms. They are calculated implicitly on the run.

Setting '''pseudo=1''' restores the old DYANA behavior with explicit pseudoatoms.

Setting '''pseudo=2''' switches to simplified pseudoatom names, such as HB instead of QB, HD1 instead of QD1, and HD instead of QQD of Leu. This is the setting to be used when reading chemical shifts from CARA. Coordinate files will contain explicit pseudoatoms, as with '''pseudo=1'''

Setting '''pseudo=3''' allows X-Plor/CNS pseudoatom names, like HX* instead of QX. For some reason using '''translate xplor''' is not enough to do the conversion for all the atoms.

 

=== The Initialization File:  init.cya ===

The init.cya is a local initialization file, which is read when cyana starts. It should be located in the directory where CYANA is run. In a given project the same file can be used for nearly all calculations.

Create your own init.cya file with the following lines in a text editor or download this template [[Media:CYANA_init.cya|init.cya]] file: 
<pre>name:=XXXX # Replace XXXX with NESG ID
nproc=2 # Number of processors on a workstation
rmsdrange:=20..72 # RMSD reported for these residues after structure calculation
# Read the standard and special libraries
cyanalib
read lib $cyanadir/lib/special.lib append
pseudo=2 # Allows HB, HD, etc. pseudoatom names, use with CARA
read seq $name # Initialize
</pre>
Replace XXXX with your NESG target ID. It is convenient to have the sequence and atomlist files named as <tt>XXXX.seq</tt> and <tt>XXXX.prot</tt>.

*<tt>nproc</tt> defines the number of processors on a workstation.
*<tt>rmsdrange</tt> is only used in structure calculation. Set the range to a valid residue range. From NOE patterns you can exclude flexible N- and C-terminal parts. If you have a flexible loop in the middle you can specify the range as <tt>10..30,40..70</tt>.
*<tt>cyanalib</tt> reads the default <tt>cyana.lib</tt> residue library. The <tt>special.lib</tt> library is appended to use non-standard residues, (i.e., His tautomers).
*<tt>pseudo=2</tt> is only necessary to read atom lists created with CARA, because they have H* for pseudoatom labels. Comment it out, or use <tt>pseudo=0</tt> if you take atom lists from XEASY. 

 

=== The Structure Calculation File:  CALC.cya ===

There are calculation demos for automatic assignment (~/demo/auto) and simple structure calculation (~/demo/manual) runs.

Here is a CALC.cya script for automatic NOE assignment: 
<pre>peaks  := c13.peaks,n15.peaks,aro.peaks # names of NOESY peak lists
prot  := demo # names of chemical shift lists
constraints := demo.aco # additional (non-NOE) constraints
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..100 # residue range for RMSD calculation
randomseed  := 434726 # random number generator seed

noeassign peaks=$peaks prot=$prot autoaco
</pre>
To prevent CYANA from changing existing peak assignments you need to define a subroutine to select the peaks to keep: 
<pre>peaks  := c13.peaks,n15.peaks,aro.peaks # names of NOESY peak lists
prot  := demo # names of chemical shift lists
constraints := demo.aco # additional (non-NOE) constraints
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..100 # residue range for RMSD calculation
randomseed  := 434726 # random number generator seed

subroutine KEEP
peaks select "*, * number=2..7999"
end

noeassign peaks=$peaks prot=$prot autoaco keep=KEEP
</pre>
Here, subroutine <tt>KEEP</tt> is used to keep the assignments for peaks with peak numbers from 2 to 7999.

 

Here is a CALC.cya script for manual structure calculation: 
<pre>peaks  := c13,n15,aro # names of peak lists
prot  := demo # names of proton lists
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)
calibration:= 6.7E5,8.2E5,8.0E4 # calibration constants (will be determined
# automatically, if commented out)
dref  := 4.2 # average upper distance limit for
# automatic calibration

if (master) then

# ---- check consistency of peak and chemical shift lists----

peakcheck peaks=$peaks prot=$prot

# ---- calibration ----

calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref
peaks calibrate "**" simple
write upl $name-in.upl
distance modify
write upl $name.upl

end if
synchronize

# ---- structure calculation ----

read seq $name.seq # re-read sequence to initialize
read upl $name.upl # read upper distance limits
read aco $name.aco # read angle constraints
seed=5671 # random number generator seed
calc_all structures=100 command=anneal steps=10000 # calculate 100 conformers
overview $name.ovw structures=20 pdb # write overview file and coordinates
</pre>
Note the order in which tolerances are given.

The '''calibration''' field can be left empty, in this case '''dref''' will be used to derive calibration constants. If '''dref''' is not specified noeassign.cya will use a default value of 4.0. During calculation noeassign.cya will also relax the calibration if needed (that is in "elastic" mode, which is the default).

'''constraints''' need not be non-NOE despite what the comment says. You can add *.aco, *.upl, *.lol, and even *.cya macros for stereospecific assignments (haven't tested it yet, but that's the way CYANA adds stereospecific assignments in the final round).

'''master''' and '''synchronize''' keywords are needed for running on a cluster.

'''peakcheck''' checks the peaklist assignments against the atom list. Always check CYANA output for '''peakcheck''' results - those huge upl violations may be caused by mis-assigned peaks.

 

=== NOE Calibration ===

CYANA 2.1 by default does not use explicit pseudoatom corrections in distance constraints. Instead, these corrections are applied implicitly on-the-fly. This behavior is turned on by setting '''expand=1'''.

Calibration is thus performed with the undocumented statement '''peaks calibrate "**" simple'''. Trivial calculations show, however, that this command uses a simple r^-6 calibration without adding pseudoatom corrections.

Old calibration macros, such as '''calibrate.cya''' and '''caliba.cya''' are still allowed, but they do add explicit pseudoatom corrections. So if want to use them, don't forget to set '''expand=0'''. Omitting it will result in applying corrections twice, making the corresponding constraints very loose.

This is, of course, a matter of huge confusion since both methods produce otherwise identical *.upl files. Be sure you know HOW you calibrate your NOEs.

To modify upper and lower distance cutoffs for NOE calibration, use '''set upl_values:=2.4,6.0'''. The defaults are 2.4 and 5.5.

For a more detailed description of NOE calibration using CYANA, follow this [[NOE Calibration Using CYANA|link]].

 

=== Dihedral Angle Constraints ===

In CYANA, dihedral angle constraints are specified in a <tt>.aco</tt> file.

Dihedral angle constraints for structure calculation in CYANA can come from a variety of sources.  For example, the [[FOUND|FOUND]] module derives dihedral angle constraints based on local NOE data.

Programs such as [[TALOS|TALOS]] provide backbone phi and psi torsion angle constraints based on chemical shifts.  In our structure determination pipeline we often make use of TALOS-derived backbone torsion angle constraints in our calculations. 

 

=== Stereospecific Assignments ===

Constraints for diastereotopic atoms (such as HB2, HB3) are treated as ambiguous by CYANA. This is switched on with '''swap=1'''.

For the manual run you may want to have '''swap=0''' to be compatible with DYANA behavior. This option is apparently not necessary when distance modification is applied.

Distance modification does not affect Phe and Tyr ring atoms HD1/2 and HE1/2. Therefore, if you have degenerate ring chemical shift (as is almost always the case) make sure you have them labeled QD and QE

External stereospecific assignments determined with [[GLOMSA|'''GLOMSA''']] or with the help of a fractionally (i.e., 5%) 13C-labeled sample [3] can be defined with a custom macro like this: 
<pre> # VAL
atom stereo "QG1 25 36 38 87"
atom stereo "QG1 43 90"
atom swap "QG1 43 90"
# LEU
atom stereo "QD1 60 63 97"
atom stereo "QD1 35 56"
atom swap "QD1 35 56"
</pre>
Here the syntax of CYANA 2.1 requires double quotes. For some strange reason, methyl groups should be written with the letter "Q" even if '''pseudo=2''' is used.

 

=== NOESY Peak Lists ===

CYANA 2.1 can produce multiple assignments for a peak. Below is a part of an aliphatic NOESY peaklist with peak #6 having two assignments. #VC tags specify the weights given to individual assignments. Calibration of this peak yields two constraints splitting the peak integral according to these weights. 
<pre># Number of dimensions 3
#FORMAT xeasy3D
#INAME 1 H
#INAME 2 C
#INAME 3 h
#CYANAFORMAT HCh
1 4.147 51.731 1.474 3 U 7.953E+03 0.000E+00 - 0 2234 2233 2238 #QU 1.000 #SUP 1.00
2 4.147 51.731 4.251 4 U 4.181E+03 0.000E+00 - 0 0 0 0
3 4.147 51.731 7.791 3 U 6.017E+03 0.000E+00 - 0 2234 2233 232 #QU 1.000 #SUP 1.00
4 1.474 22.186 0.515 3 U 1.481E+03 0.000E+00 - 0 2390 2389 1417 #QU 0.981 #SUP 0.98
5 1.474 22.186 1.249 3 U 2.610E+04 0.000E+00 - 0 2390 2389 1706 #QU 0.987 #SUP 0.99
6 1.474 22.186 2.635 3 U 1.396E+03 0.000E+00 - 0 2390 2389 1715 #VC 0.47897 #QU 0.774 #SUP 0.96
2390 2389 1815 #VC 0.52103 #QU 0.813 #SUP 0.96
7 1.474 22.186 3.863 3 U 1.418E+04 0.000E+00 - 0 2390 2389 1657 #QU 0.885 #SUP 0.88
8 1.474 22.186 4.147 3 U 1.448E+04 0.000E+00 - 0 2238 2237 2234 #QU 1.000 #SUP 1.00
</pre>
The peaklists produced by CYANA 2.1 are not backwards-compatible with XEASY, but there are Lua scripts, which can read them into CARA including the information on ambiguous assignments. UBNMR should also be able to handle them in the future.

When supplying completely unassigned peaks for automatic NOE assignment it is necessary to include a line like '''#CYANAFORMAT HCh''' in the header.

 

== '''CYANA 3.0''' ==

Again please consult the [http://www.cyana.org/wiki/index.php/CYANA_3.0_Reference_Manual CYANA 3.0 wiki] for complete details on file formats, input files for CYANA, and other documentation.

=== Residue Library ===

A [http://www.cyana.org/wiki/index.php/Residue_library_file residue library] defines all properties of a residue including atom types, the nomenclature, the dihedral angle definitions, the covalent connectivities and the standard geometry. The standard geometry of the ECEPP/2 force field [4,5] is used for all amino acid residue types.  Standard residues are collected in the '''cyana.lib''' library; special residue types are in the '''special.lib''' library.

=== Sequence File ===

The [http://www.cyana.org/wiki/index.php/Sequence_file sequence file] (.seq) defines the sequence of the molecule you are working with.  Special residue types (i.e., oxdized cysteine, histidine tautomers, and cis-peptide bonds) can also be defined in the sequence file as follows:

*oxidized cysteine:  CISS
*charged histidine:  HIS+
*Nε2H neutral tautomer:  HIST (the default HIS specifies the Nδ1H neutral tautomer).
*cis-peptide bond:  place a "c" before the residue name;  i.e., cPRO
*invisible intermolecular linkers:  PL, LL, LL2, LL5, LP

 

=== Automated Structure calculation ===

<pre>
peaks := n,ali,aro # names of NOESY peak lists
prot := $name # names of chemical shift lists
restraints := talos.aco,stereo.cya # additional (non-NOE) constraints
tolerance := 0.04,0.02,0.4 # chemical shift tolerances
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)
upl_values := 2.4,5.5 # calibration cutoffs
cut_upl=0.05
calibration_constant:= # NOE calibration parameters
structures := 100,20 # number of initial, final structures
steps := 10000 # number of torsion angle dynamics steps
rmsdrange := 20..102 # residue range for RMSD calculation
randomseed := 562 # random number generator seed
calibration_dref := 4.0 # average distance for calibration, default 4.0
keep := # set to KEEP to retain existing assignments

weight_rdc = 0.002 # weight for RDC restraints
cut_rdc = 1.0 # cut-off for RDC violations
opt_tensor = 1 # alignment tensor optimization

subroutine KEEP
peaks select "*,*"
end

noeassign peaks=$peaks prot=$prot keep=$keep autoaco
</pre>

=== '''A Simple Automated Structure Calculation Using CYANA 3.0''' ===

This section provides an example of a standard automatic NOESY assignment calculation using CYANA 3.0 on a monomeric protein.

==== Input Files ====

Collect the following files in your directory (see the attached files for examples and formatting): 

*[[Media:CYANA30ex_init.cya|init.cya]]:  initialization file. Defines the protein name (i.e., PROT), residue library(ies), number of processors used, and rmsd residue range.
*[[Media:CYANA3ex_CALC.cya|CALC.cya]]:  structure calculation file: Defines the peak lists, tolerances, any NOE calibration parameters (default is automatic calibration), total number of structures calculated in each cycle, number of structures with lowest target function retained after each cycle, number of torsion angle dynamics steps, random seed.
*[[Media:CYANA3ex_PfR193A.seq|PROT.seq]]:  protein sequence file.
*[[Media:CYANA3ex_PfR193A.aco|PROT.aco]]:  dihedral angle constraint file.
*[[Media:CYANA3ex_PfR193A_h2o.prot|filename.prot]]:  chemical shift assignment list.  You should make all degenerate geminal proton assignments Q's, as well as degenerate side chain aromatics (HD1/HD2 and HE1/HE2).
**if your assignments are in a bmrb file (2.1), start cyana, read in the bmrb file, and then write the shifts out to a prot file as follows:
<pre> Open project in cyana 3.0:
read bmrb [finename.bmrb]
write prot [filename.prot]</pre>
*[[Media:CYANA3ex_PfR193A_n15.peaks|filename1.peaks]], [[Media:CYANA3ex_PfR193A_c13_h2o_ali.peaks|filename2.peaks]], [[Media:CYANA3ex_PfR193A_c13_h2o_aro.peaks|filename3.peaks]]:  NOESY peak lists in XEASY format.  The peak lists are unassigned.
*[[Media:CYANA3ex_ssa.cya|ssa.cya]]:  file with stereospecific assignments defined.
*other:  other constraint files such as, manual upper and lower distance limits, hydrogen bond constraints.

==== Running the Program ====

To run CYANA 3.0 on our cluster at CABM, login to master3 and type:
<pre> /farm/software/cyana3.0/bin/cyana CALC > log.out
</pre>
==== Output Files ====

A CYANA structure calculation run will produce .pdb, .upl, .noa and .ovw files for each cycle and the final cycle, as well as log and ramachandran files for the run.

The command <tt>cyanatable</tt> produces a summary table of an automated NOE assignment structure calculation run.

 

== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/9367762?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2 1.    Güntert, P,, Mumenthaler, C. and Wüthrich, K. (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. ''J. Mol. Biol. 273'', 283-298.]

[http://www.ncbi.nlm.nih.gov/pubmed/12051947?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=5 2.    Herrmann, T., Güntert P. and Wüthrich, K. (2002) Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA.  ''J. Mol. Biol. 319'' , 209-227.] 

[http://www.ncbi.nlm.nih.gov/pubmed/2692701?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=9 3.    Neri, D., Szyperski, T., Otting, G., Senn, H. and Wüthrich, K. (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. ''Biochemistry 28'', 7510-7516.]

4.    Momany, F.A., McGuire, R.F., Burgess, A.W. and Scheraga, H.A. (1975)  Energy parameters in polypeptides. VII. Geometric parameters, partial atomic charges, nonbonded interactions, hydrogen bond interactions, and intrinsic torsional potentials for the naturally occurring amino acids.  ''J. Phys. Chem. 79'', 2361-2381.

5.    Nemethy, G., Pottle, M.S. and Scheraga, H.A. (1983)  Energy parameters in polypeptides. 9. Updating of geometrical parameters, nonbonded interactions, and hydrogen bond interactions for the naturally occurring amino acids.  ''J. Phys. Chem. 87'', 1883-1887.

CYANA

2011-10-24T21:41:58Z

Alex: /* Automated Structure calculation */

== '''Introduction''' ==

CYANA is a macromolecular structure calculation algorithm based on simulated annealing molecular dynamics calculations in torsional angle space, in contrast to Cartesian space [1,2].  Here the only degrees of freedom are torsion angles with covalent structure parameters kept fixed, thereby significantly decreasing the number of degrees of freedom in the calculation.

The current version of CYANA is 3.0, and it is capable of handling orientational (i.e., RDC) constraints.  There is now a nice [http://www.cyana.org/wiki/index.php/CYANA_3.0_Reference_Manual CYANA 3.0 wiki] with file explanations, tutorials and theory.

In the sections below we describe input files and protocols used in the NESG for both CYANA 2.1 and 3.0.

 

== '''CYANA 2.1''' ==

=== Residue Library ===

CYANA 2.1 uses a new library ~/lib/cyana.lib. It is loaded by the <tt>cyanalib</tt> command. For back-compatibility there is also the old DYANA library dyana.lib (loaded with <tt>dyanalib</tt>, of course).

The main difference is larger van der Waals radii. This will give you a larger target function than DYANA, but also better clash scores.

Residue nomenclature is also different - there are no charged species, like ARG+ or GLU-.

 

=== Atom Nomenclature ===

Atom nomenclature was made compatible with BMRB standard. The deviations from XEASY/DYANA conventions are: HN <-> H, HA1 <-> HA2 and HA2 <-> HA3 for GLY.

There a is macro '''translate.cya''', which is used to convert input to different formats. For example, to read files with DYANA nomenclature, enter <tt>translate dyana</tt>. To switch back to CYANA 2.1 convention type <tt>translate off</tt>.

 

=== Pseudoatom Treatment ===

Pseudoatom handling is switched by setting '''pseudo=x''', where x is 0, 1, 2, or 3.

With '''pseudo=0''', the default setting, coordinate files *.cor and *.pdb do not contain pseudoatoms. They are calculated implicitly on the run.

Setting '''pseudo=1''' restores the old DYANA behavior with explicit pseudoatoms.

Setting '''pseudo=2''' switches to simplified pseudoatom names, such as HB instead of QB, HD1 instead of QD1, and HD instead of QQD of Leu. This is the setting to be used when reading chemical shifts from CARA. Coordinate files will contain explicit pseudoatoms, as with '''pseudo=1'''

Setting '''pseudo=3''' allows X-Plor/CNS pseudoatom names, like HX* instead of QX. For some reason using '''translate xplor''' is not enough to do the conversion for all the atoms.

 

=== The Initialization File:  init.cya ===

The init.cya is a local initialization file, which is read when cyana starts. It should be located in the directory where CYANA is run. In a given project the same file can be used for nearly all calculations.

Create your own init.cya file with the following lines in a text editor or download this template [[Media:CYANA_init.cya|init.cya]] file: 
<pre>name:=XXXX # Replace XXXX with NESG ID
nproc=2 # Number of processors on a workstation
rmsdrange:=20..72 # RMSD reported for these residues after structure calculation
# Read the standard and special libraries
cyanalib
read lib $cyanadir/lib/special.lib append
pseudo=2 # Allows HB, HD, etc. pseudoatom names, use with CARA
read seq $name # Initialize
</pre>
Replace XXXX with your NESG target ID. It is convenient to have the sequence and atomlist files named as <tt>XXXX.seq</tt> and <tt>XXXX.prot</tt>.

*<tt>nproc</tt> defines the number of processors on a workstation.
*<tt>rmsdrange</tt> is only used in structure calculation. Set the range to a valid residue range. From NOE patterns you can exclude flexible N- and C-terminal parts. If you have a flexible loop in the middle you can specify the range as <tt>10..30,40..70</tt>.
*<tt>cyanalib</tt> reads the default <tt>cyana.lib</tt> residue library. The <tt>special.lib</tt> library is appended to use non-standard residues, (i.e., His tautomers).
*<tt>pseudo=2</tt> is only necessary to read atom lists created with CARA, because they have H* for pseudoatom labels. Comment it out, or use <tt>pseudo=0</tt> if you take atom lists from XEASY. 

 

=== The Structure Calculation File:  CALC.cya ===

There are calculation demos for automatic assignment (~/demo/auto) and simple structure calculation (~/demo/manual) runs.

Here is a CALC.cya script for automatic NOE assignment: 
<pre>peaks  := c13.peaks,n15.peaks,aro.peaks # names of NOESY peak lists
prot  := demo # names of chemical shift lists
constraints := demo.aco # additional (non-NOE) constraints
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..100 # residue range for RMSD calculation
randomseed  := 434726 # random number generator seed

noeassign peaks=$peaks prot=$prot autoaco
</pre>
To prevent CYANA from changing existing peak assignments you need to define a subroutine to select the peaks to keep: 
<pre>peaks  := c13.peaks,n15.peaks,aro.peaks # names of NOESY peak lists
prot  := demo # names of chemical shift lists
constraints := demo.aco # additional (non-NOE) constraints
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..100 # residue range for RMSD calculation
randomseed  := 434726 # random number generator seed

subroutine KEEP
peaks select "*, * number=2..7999"
end

noeassign peaks=$peaks prot=$prot autoaco keep=KEEP
</pre>
Here, subroutine <tt>KEEP</tt> is used to keep the assignments for peaks with peak numbers from 2 to 7999.

 

Here is a CALC.cya script for manual structure calculation: 
<pre>peaks  := c13,n15,aro # names of peak lists
prot  := demo # names of proton lists
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)
calibration:= 6.7E5,8.2E5,8.0E4 # calibration constants (will be determined
# automatically, if commented out)
dref  := 4.2 # average upper distance limit for
# automatic calibration

if (master) then

# ---- check consistency of peak and chemical shift lists----

peakcheck peaks=$peaks prot=$prot

# ---- calibration ----

calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref
peaks calibrate "**" simple
write upl $name-in.upl
distance modify
write upl $name.upl

end if
synchronize

# ---- structure calculation ----

read seq $name.seq # re-read sequence to initialize
read upl $name.upl # read upper distance limits
read aco $name.aco # read angle constraints
seed=5671 # random number generator seed
calc_all structures=100 command=anneal steps=10000 # calculate 100 conformers
overview $name.ovw structures=20 pdb # write overview file and coordinates
</pre>
Note the order in which tolerances are given.

The '''calibration''' field can be left empty, in this case '''dref''' will be used to derive calibration constants. If '''dref''' is not specified noeassign.cya will use a default value of 4.0. During calculation noeassign.cya will also relax the calibration if needed (that is in "elastic" mode, which is the default).

'''constraints''' need not be non-NOE despite what the comment says. You can add *.aco, *.upl, *.lol, and even *.cya macros for stereospecific assignments (haven't tested it yet, but that's the way CYANA adds stereospecific assignments in the final round).

'''master''' and '''synchronize''' keywords are needed for running on a cluster.

'''peakcheck''' checks the peaklist assignments against the atom list. Always check CYANA output for '''peakcheck''' results - those huge upl violations may be caused by mis-assigned peaks.

 

=== NOE Calibration ===

CYANA 2.1 by default does not use explicit pseudoatom corrections in distance constraints. Instead, these corrections are applied implicitly on-the-fly. This behavior is turned on by setting '''expand=1'''.

Calibration is thus performed with the undocumented statement '''peaks calibrate "**" simple'''. Trivial calculations show, however, that this command uses a simple r^-6 calibration without adding pseudoatom corrections.

Old calibration macros, such as '''calibrate.cya''' and '''caliba.cya''' are still allowed, but they do add explicit pseudoatom corrections. So if want to use them, don't forget to set '''expand=0'''. Omitting it will result in applying corrections twice, making the corresponding constraints very loose.

This is, of course, a matter of huge confusion since both methods produce otherwise identical *.upl files. Be sure you know HOW you calibrate your NOEs.

To modify upper and lower distance cutoffs for NOE calibration, use '''set upl_values:=2.4,6.0'''. The defaults are 2.4 and 5.5.

For a more detailed description of NOE calibration using CYANA, follow this [[NOE Calibration Using CYANA|link]].

 

=== Dihedral Angle Constraints ===

In CYANA, dihedral angle constraints are specified in a <tt>.aco</tt> file.

Dihedral angle constraints for structure calculation in CYANA can come from a variety of sources.  For example, the [[FOUND|FOUND]] module derives dihedral angle constraints based on local NOE data.

Programs such as [[TALOS|TALOS]] provide backbone phi and psi torsion angle constraints based on chemical shifts.  In our structure determination pipeline we often make use of TALOS-derived backbone torsion angle constraints in our calculations. 

 

=== Stereospecific Assignments ===

Constraints for diastereotopic atoms (such as HB2, HB3) are treated as ambiguous by CYANA. This is switched on with '''swap=1'''.

For the manual run you may want to have '''swap=0''' to be compatible with DYANA behavior. This option is apparently not necessary when distance modification is applied.

Distance modification does not affect Phe and Tyr ring atoms HD1/2 and HE1/2. Therefore, if you have degenerate ring chemical shift (as is almost always the case) make sure you have them labeled QD and QE

External stereospecific assignments determined with [[GLOMSA|'''GLOMSA''']] or with the help of a fractionally (i.e., 5%) 13C-labeled sample [3] can be defined with a custom macro like this: 
<pre> # VAL
atom stereo "QG1 25 36 38 87"
atom stereo "QG1 43 90"
atom swap "QG1 43 90"
# LEU
atom stereo "QD1 60 63 97"
atom stereo "QD1 35 56"
atom swap "QD1 35 56"
</pre>
Here the syntax of CYANA 2.1 requires double quotes. For some strange reason, methyl groups should be written with the letter "Q" even if '''pseudo=2''' is used.

 

=== NOESY Peak Lists ===

CYANA 2.1 can produce multiple assignments for a peak. Below is a part of an aliphatic NOESY peaklist with peak #6 having two assignments. #VC tags specify the weights given to individual assignments. Calibration of this peak yields two constraints splitting the peak integral according to these weights. 
<pre># Number of dimensions 3
#FORMAT xeasy3D
#INAME 1 H
#INAME 2 C
#INAME 3 h
#CYANAFORMAT HCh
1 4.147 51.731 1.474 3 U 7.953E+03 0.000E+00 - 0 2234 2233 2238 #QU 1.000 #SUP 1.00
2 4.147 51.731 4.251 4 U 4.181E+03 0.000E+00 - 0 0 0 0
3 4.147 51.731 7.791 3 U 6.017E+03 0.000E+00 - 0 2234 2233 232 #QU 1.000 #SUP 1.00
4 1.474 22.186 0.515 3 U 1.481E+03 0.000E+00 - 0 2390 2389 1417 #QU 0.981 #SUP 0.98
5 1.474 22.186 1.249 3 U 2.610E+04 0.000E+00 - 0 2390 2389 1706 #QU 0.987 #SUP 0.99
6 1.474 22.186 2.635 3 U 1.396E+03 0.000E+00 - 0 2390 2389 1715 #VC 0.47897 #QU 0.774 #SUP 0.96
2390 2389 1815 #VC 0.52103 #QU 0.813 #SUP 0.96
7 1.474 22.186 3.863 3 U 1.418E+04 0.000E+00 - 0 2390 2389 1657 #QU 0.885 #SUP 0.88
8 1.474 22.186 4.147 3 U 1.448E+04 0.000E+00 - 0 2238 2237 2234 #QU 1.000 #SUP 1.00
</pre>
The peaklists produced by CYANA 2.1 are not backwards-compatible with XEASY, but there are Lua scripts, which can read them into CARA including the information on ambiguous assignments. UBNMR should also be able to handle them in the future.

When supplying completely unassigned peaks for automatic NOE assignment it is necessary to include a line like '''#CYANAFORMAT HCh''' in the header.

 

== '''CYANA 3.0''' ==

Again please consult the [http://www.cyana.org/wiki/index.php/CYANA_3.0_Reference_Manual CYANA 3.0 wiki] for complete details on file formats, input files for CYANA, and other documentation.

=== Residue Library ===

A [http://www.cyana.org/wiki/index.php/Residue_library_file residue library] defines all properties of a residue including atom types, the nomenclature, the dihedral angle definitions, the covalent connectivities and the standard geometry. The standard geometry of the ECEPP/2 force field [4,5] is used for all amino acid residue types.  Standard residues are collected in the '''cyana.lib''' library; special residue types are in the '''special.lib''' library.

=== Sequence File ===

The [http://www.cyana.org/wiki/index.php/Sequence_file sequence file] (.seq) defines the sequence of the molecule you are working with.  Special residue types (i.e., oxdized cysteine, histidine tautomers, and cis-peptide bonds) can also be defined in the sequence file as follows:

*oxidized cysteine:  CISS
*charged histidine:  HIS+
*Nε2H neutral tautomer:  HIST (the default HIS specifies the Nδ1H neutral tautomer).
*cis-peptide bond:  place a "c" before the residue name;  i.e., cPRO
*invisible intermolecular linkers:  PL, LL, LL2, LL5, LP

 

=== Automated Structure calculation ===

<pre>
peaks := n,ali,aro # names of NOESY peak lists
prot := $name # names of chemical shift lists
restraints := talos.aco,stereo.cya # additional (non-NOE) constraints
tolerance := 0.04,0.02,0.4 # chemical shift tolerances
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)
upl_values := 2.4,5.5 # calibration cutoffs
cut_upl=0.05
calibration_constant:= # NOE calibration parameters
structures := 100,20 # number of initial, final structures
steps := 10000 # number of torsion angle dynamics steps
rmsdrange := 20..102 # residue range for RMSD calculation
randomseed := 562 # random number generator seed
calibration_dref := 4.0 # average distance for calibration, default 4.0
keep := # set to KEEP to retain existing assignments

weight_rdc = 0.002 # weight for RDC restraints
cut_rdc = 1.0 # cut-off for RDC violations
opt_tensor = 1 # alignment tensor optimization

subroutine KEEP
peaks select "*,*"
end

noeassign peaks=$peaks prot=$prot keep=$keep autoaco
</pre>

=== '''A Simple Automated Structure Calculation Using CYANA 3.0''' ===

This section provides an example of a standard automatic NOESY assignment calculation using CYANA 3.0 on a monomeric protein.

==== Input Files ====

Collect the following files in your directory (see the attached files for examples and formatting): 

*[[Media:CYANA30ex_init.cya|init.cya]]:  initialization file. Defines the protein name (i.e., PROT), residue library(ies), number of processors used, and rmsd residue range.
*[[Media:CYANA3ex_CALC.cya|CALC.cya]]:  structure calculation file: Defines the peak lists, tolerances, any NOE calibration parameters (default is automatic calibration), total number of structures calculated in each cycle, number of structures with lowest target function retained after each cycle, number of torsion angle dynamics steps, random seed.
*[[Media:CYANA3ex_PfR193A.seq|PROT.seq]]:  protein sequence file.
*[[Media:CYANA3ex_PfR193A.aco|PROT.aco]]:  dihedral angle constraint file.
*[[Media:CYANA3ex_PfR193A_h2o.prot|filename.prot]]:  chemical shift assignment list.  You should make all degenerate geminal proton assignments Q's, as well as degenerate side chain aromatics (HD1/HD2 and HE1/HE2).
**if your assignments are in a bmrb file (2.1), start cyana, read in the bmrb file, and then write the shifts out to a prot file as follows:
<pre> Open project in cyana 3.0:
read bmrb [finename.bmrb]
write prot [filename.prot]</pre>
*[[Media:CYANA3ex_PfR193A_n15.peaks|filename1.peaks]], [[Media:CYANA3ex_PfR193A_c13_h2o_ali.peaks|filename2.peaks]], [[Media:CYANA3ex_PfR193A_c13_h2o_aro.peaks|filename3.peaks]]:  NOESY peak lists in XEASY format.  The peak lists are unassigned.
*[[Media:CYANA3ex_ssa.cya|ssa.cya]]:  file with stereospecific assignments defined.
*other:  other constraint files such as, manual upper and lower distance limits, hydrogen bond constraints.

==== Running the Program ====

To run CYANA 3.0 on our cluster at CABM, login to master3 and type:
<pre> /farm/software/cyana3.0/bin/cyana CALC > log.out
</pre>
==== Output Files ====

A CYANA structure calculation run will produce .pdb, .upl, .noa and .ovw files for each cycle and the final cycle, as well as log and ramachandran files for the run.

The command <tt>cyanatable</tt> produces a summary table of an automated NOE assignment structure calculation run.

 

== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/9367762?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2 1.    Güntert, P,, Mumenthaler, C. and Wüthrich, K. (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. ''J. Mol. Biol. 273'', 283-298.]

[http://www.ncbi.nlm.nih.gov/pubmed/12051947?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=5 2.    Herrmann, T., Güntert P. and Wüthrich, K. (2002) Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA.  ''J. Mol. Biol. 319'' , 209-227.] 

[http://www.ncbi.nlm.nih.gov/pubmed/2692701?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=9 3.    Neri, D., Szyperski, T., Otting, G., Senn, H. and Wüthrich, K. (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. ''Biochemistry 28'', 7510-7516.]

4.    Momany, F.A., McGuire, R.F., Burgess, A.W. and Scheraga, H.A. (1975)  Energy parameters in polypeptides. VII. Geometric parameters, partial atomic charges, nonbonded interactions, hydrogen bond interactions, and intrinsic torsional potentials for the naturally occurring amino acids.  ''J. Phys. Chem. 79'', 2361-2381.

5.    Nemethy, G., Pottle, M.S. and Scheraga, H.A. (1983)  Energy parameters in polypeptides. 9. Updating of geometrical parameters, nonbonded interactions, and hydrogen bond interactions for the naturally occurring amino acids.  ''J. Phys. Chem. 87'', 1883-1887.

CYANA

2011-10-24T21:41:48Z

Alex: /* Automated Structure calculation */

== '''Introduction''' ==

CYANA is a macromolecular structure calculation algorithm based on simulated annealing molecular dynamics calculations in torsional angle space, in contrast to Cartesian space [1,2].  Here the only degrees of freedom are torsion angles with covalent structure parameters kept fixed, thereby significantly decreasing the number of degrees of freedom in the calculation.

The current version of CYANA is 3.0, and it is capable of handling orientational (i.e., RDC) constraints.  There is now a nice [http://www.cyana.org/wiki/index.php/CYANA_3.0_Reference_Manual CYANA 3.0 wiki] with file explanations, tutorials and theory.

In the sections below we describe input files and protocols used in the NESG for both CYANA 2.1 and 3.0.

 

== '''CYANA 2.1''' ==

=== Residue Library ===

CYANA 2.1 uses a new library ~/lib/cyana.lib. It is loaded by the <tt>cyanalib</tt> command. For back-compatibility there is also the old DYANA library dyana.lib (loaded with <tt>dyanalib</tt>, of course).

The main difference is larger van der Waals radii. This will give you a larger target function than DYANA, but also better clash scores.

Residue nomenclature is also different - there are no charged species, like ARG+ or GLU-.

 

=== Atom Nomenclature ===

Atom nomenclature was made compatible with BMRB standard. The deviations from XEASY/DYANA conventions are: HN <-> H, HA1 <-> HA2 and HA2 <-> HA3 for GLY.

There a is macro '''translate.cya''', which is used to convert input to different formats. For example, to read files with DYANA nomenclature, enter <tt>translate dyana</tt>. To switch back to CYANA 2.1 convention type <tt>translate off</tt>.

 

=== Pseudoatom Treatment ===

Pseudoatom handling is switched by setting '''pseudo=x''', where x is 0, 1, 2, or 3.

With '''pseudo=0''', the default setting, coordinate files *.cor and *.pdb do not contain pseudoatoms. They are calculated implicitly on the run.

Setting '''pseudo=1''' restores the old DYANA behavior with explicit pseudoatoms.

Setting '''pseudo=2''' switches to simplified pseudoatom names, such as HB instead of QB, HD1 instead of QD1, and HD instead of QQD of Leu. This is the setting to be used when reading chemical shifts from CARA. Coordinate files will contain explicit pseudoatoms, as with '''pseudo=1'''

Setting '''pseudo=3''' allows X-Plor/CNS pseudoatom names, like HX* instead of QX. For some reason using '''translate xplor''' is not enough to do the conversion for all the atoms.

 

=== The Initialization File:  init.cya ===

The init.cya is a local initialization file, which is read when cyana starts. It should be located in the directory where CYANA is run. In a given project the same file can be used for nearly all calculations.

Create your own init.cya file with the following lines in a text editor or download this template [[Media:CYANA_init.cya|init.cya]] file: 
<pre>name:=XXXX # Replace XXXX with NESG ID
nproc=2 # Number of processors on a workstation
rmsdrange:=20..72 # RMSD reported for these residues after structure calculation
# Read the standard and special libraries
cyanalib
read lib $cyanadir/lib/special.lib append
pseudo=2 # Allows HB, HD, etc. pseudoatom names, use with CARA
read seq $name # Initialize
</pre>
Replace XXXX with your NESG target ID. It is convenient to have the sequence and atomlist files named as <tt>XXXX.seq</tt> and <tt>XXXX.prot</tt>.

*<tt>nproc</tt> defines the number of processors on a workstation.
*<tt>rmsdrange</tt> is only used in structure calculation. Set the range to a valid residue range. From NOE patterns you can exclude flexible N- and C-terminal parts. If you have a flexible loop in the middle you can specify the range as <tt>10..30,40..70</tt>.
*<tt>cyanalib</tt> reads the default <tt>cyana.lib</tt> residue library. The <tt>special.lib</tt> library is appended to use non-standard residues, (i.e., His tautomers).
*<tt>pseudo=2</tt> is only necessary to read atom lists created with CARA, because they have H* for pseudoatom labels. Comment it out, or use <tt>pseudo=0</tt> if you take atom lists from XEASY. 

 

=== The Structure Calculation File:  CALC.cya ===

There are calculation demos for automatic assignment (~/demo/auto) and simple structure calculation (~/demo/manual) runs.

Here is a CALC.cya script for automatic NOE assignment: 
<pre>peaks  := c13.peaks,n15.peaks,aro.peaks # names of NOESY peak lists
prot  := demo # names of chemical shift lists
constraints := demo.aco # additional (non-NOE) constraints
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..100 # residue range for RMSD calculation
randomseed  := 434726 # random number generator seed

noeassign peaks=$peaks prot=$prot autoaco
</pre>
To prevent CYANA from changing existing peak assignments you need to define a subroutine to select the peaks to keep: 
<pre>peaks  := c13.peaks,n15.peaks,aro.peaks # names of NOESY peak lists
prot  := demo # names of chemical shift lists
constraints := demo.aco # additional (non-NOE) constraints
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..100 # residue range for RMSD calculation
randomseed  := 434726 # random number generator seed

subroutine KEEP
peaks select "*, * number=2..7999"
end

noeassign peaks=$peaks prot=$prot autoaco keep=KEEP
</pre>
Here, subroutine <tt>KEEP</tt> is used to keep the assignments for peaks with peak numbers from 2 to 7999.

 

Here is a CALC.cya script for manual structure calculation: 
<pre>peaks  := c13,n15,aro # names of peak lists
prot  := demo # names of proton lists
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)
calibration:= 6.7E5,8.2E5,8.0E4 # calibration constants (will be determined
# automatically, if commented out)
dref  := 4.2 # average upper distance limit for
# automatic calibration

if (master) then

# ---- check consistency of peak and chemical shift lists----

peakcheck peaks=$peaks prot=$prot

# ---- calibration ----

calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref
peaks calibrate "**" simple
write upl $name-in.upl
distance modify
write upl $name.upl

end if
synchronize

# ---- structure calculation ----

read seq $name.seq # re-read sequence to initialize
read upl $name.upl # read upper distance limits
read aco $name.aco # read angle constraints
seed=5671 # random number generator seed
calc_all structures=100 command=anneal steps=10000 # calculate 100 conformers
overview $name.ovw structures=20 pdb # write overview file and coordinates
</pre>
Note the order in which tolerances are given.

The '''calibration''' field can be left empty, in this case '''dref''' will be used to derive calibration constants. If '''dref''' is not specified noeassign.cya will use a default value of 4.0. During calculation noeassign.cya will also relax the calibration if needed (that is in "elastic" mode, which is the default).

'''constraints''' need not be non-NOE despite what the comment says. You can add *.aco, *.upl, *.lol, and even *.cya macros for stereospecific assignments (haven't tested it yet, but that's the way CYANA adds stereospecific assignments in the final round).

'''master''' and '''synchronize''' keywords are needed for running on a cluster.

'''peakcheck''' checks the peaklist assignments against the atom list. Always check CYANA output for '''peakcheck''' results - those huge upl violations may be caused by mis-assigned peaks.

 

=== NOE Calibration ===

CYANA 2.1 by default does not use explicit pseudoatom corrections in distance constraints. Instead, these corrections are applied implicitly on-the-fly. This behavior is turned on by setting '''expand=1'''.

Calibration is thus performed with the undocumented statement '''peaks calibrate "**" simple'''. Trivial calculations show, however, that this command uses a simple r^-6 calibration without adding pseudoatom corrections.

Old calibration macros, such as '''calibrate.cya''' and '''caliba.cya''' are still allowed, but they do add explicit pseudoatom corrections. So if want to use them, don't forget to set '''expand=0'''. Omitting it will result in applying corrections twice, making the corresponding constraints very loose.

This is, of course, a matter of huge confusion since both methods produce otherwise identical *.upl files. Be sure you know HOW you calibrate your NOEs.

To modify upper and lower distance cutoffs for NOE calibration, use '''set upl_values:=2.4,6.0'''. The defaults are 2.4 and 5.5.

For a more detailed description of NOE calibration using CYANA, follow this [[NOE Calibration Using CYANA|link]].

 

=== Dihedral Angle Constraints ===

In CYANA, dihedral angle constraints are specified in a <tt>.aco</tt> file.

Dihedral angle constraints for structure calculation in CYANA can come from a variety of sources.  For example, the [[FOUND|FOUND]] module derives dihedral angle constraints based on local NOE data.

Programs such as [[TALOS|TALOS]] provide backbone phi and psi torsion angle constraints based on chemical shifts.  In our structure determination pipeline we often make use of TALOS-derived backbone torsion angle constraints in our calculations. 

 

=== Stereospecific Assignments ===

Constraints for diastereotopic atoms (such as HB2, HB3) are treated as ambiguous by CYANA. This is switched on with '''swap=1'''.

For the manual run you may want to have '''swap=0''' to be compatible with DYANA behavior. This option is apparently not necessary when distance modification is applied.

Distance modification does not affect Phe and Tyr ring atoms HD1/2 and HE1/2. Therefore, if you have degenerate ring chemical shift (as is almost always the case) make sure you have them labeled QD and QE

External stereospecific assignments determined with [[GLOMSA|'''GLOMSA''']] or with the help of a fractionally (i.e., 5%) 13C-labeled sample [3] can be defined with a custom macro like this: 
<pre> # VAL
atom stereo "QG1 25 36 38 87"
atom stereo "QG1 43 90"
atom swap "QG1 43 90"
# LEU
atom stereo "QD1 60 63 97"
atom stereo "QD1 35 56"
atom swap "QD1 35 56"
</pre>
Here the syntax of CYANA 2.1 requires double quotes. For some strange reason, methyl groups should be written with the letter "Q" even if '''pseudo=2''' is used.

 

=== NOESY Peak Lists ===

CYANA 2.1 can produce multiple assignments for a peak. Below is a part of an aliphatic NOESY peaklist with peak #6 having two assignments. #VC tags specify the weights given to individual assignments. Calibration of this peak yields two constraints splitting the peak integral according to these weights. 
<pre># Number of dimensions 3
#FORMAT xeasy3D
#INAME 1 H
#INAME 2 C
#INAME 3 h
#CYANAFORMAT HCh
1 4.147 51.731 1.474 3 U 7.953E+03 0.000E+00 - 0 2234 2233 2238 #QU 1.000 #SUP 1.00
2 4.147 51.731 4.251 4 U 4.181E+03 0.000E+00 - 0 0 0 0
3 4.147 51.731 7.791 3 U 6.017E+03 0.000E+00 - 0 2234 2233 232 #QU 1.000 #SUP 1.00
4 1.474 22.186 0.515 3 U 1.481E+03 0.000E+00 - 0 2390 2389 1417 #QU 0.981 #SUP 0.98
5 1.474 22.186 1.249 3 U 2.610E+04 0.000E+00 - 0 2390 2389 1706 #QU 0.987 #SUP 0.99
6 1.474 22.186 2.635 3 U 1.396E+03 0.000E+00 - 0 2390 2389 1715 #VC 0.47897 #QU 0.774 #SUP 0.96
2390 2389 1815 #VC 0.52103 #QU 0.813 #SUP 0.96
7 1.474 22.186 3.863 3 U 1.418E+04 0.000E+00 - 0 2390 2389 1657 #QU 0.885 #SUP 0.88
8 1.474 22.186 4.147 3 U 1.448E+04 0.000E+00 - 0 2238 2237 2234 #QU 1.000 #SUP 1.00
</pre>
The peaklists produced by CYANA 2.1 are not backwards-compatible with XEASY, but there are Lua scripts, which can read them into CARA including the information on ambiguous assignments. UBNMR should also be able to handle them in the future.

When supplying completely unassigned peaks for automatic NOE assignment it is necessary to include a line like '''#CYANAFORMAT HCh''' in the header.

 

== '''CYANA 3.0''' ==

Again please consult the [http://www.cyana.org/wiki/index.php/CYANA_3.0_Reference_Manual CYANA 3.0 wiki] for complete details on file formats, input files for CYANA, and other documentation.

=== Residue Library ===

A [http://www.cyana.org/wiki/index.php/Residue_library_file residue library] defines all properties of a residue including atom types, the nomenclature, the dihedral angle definitions, the covalent connectivities and the standard geometry. The standard geometry of the ECEPP/2 force field [4,5] is used for all amino acid residue types.  Standard residues are collected in the '''cyana.lib''' library; special residue types are in the '''special.lib''' library.

=== Sequence File ===

The [http://www.cyana.org/wiki/index.php/Sequence_file sequence file] (.seq) defines the sequence of the molecule you are working with.  Special residue types (i.e., oxdized cysteine, histidine tautomers, and cis-peptide bonds) can also be defined in the sequence file as follows:

*oxidized cysteine:  CISS
*charged histidine:  HIS+
*Nε2H neutral tautomer:  HIST (the default HIS specifies the Nδ1H neutral tautomer).
*cis-peptide bond:  place a "c" before the residue name;  i.e., cPRO
*invisible intermolecular linkers:  PL, LL, LL2, LL5, LP

 

=== Automated Structure calculation ===

<pre>
peaks := n,ali,aro # names of NOESY peak lists
prot := $name # names of chemical shift lists
restraints := talos.aco,stereo.cya # additional (non-NOE) constraints
tolerance := 0.04,0.02,0.4 # chemical shift tolerances
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)
upl_values := 2.4,5.5 # calibration cutoffs
cut_upl=0.05
calibration_constant:= # NOE calibration parameters
structures := 100,20 # number of initial, final structures
steps := 10000 # number of torsion angle dynamics steps
rmsdrange := 20..102 # residue range for RMSD calculation
randomseed := 562 # random number generator seed
calibration_dref := 4.0 # average distance for calibration, default 4.0
keep := # set to KEEP to retain existing assignments

weight_rdc = 0.002 # weight for RDC restraints
cut_rdc = 1.0 # cut-off for RDC violations
opt_tensor = 1 # alignment tensor optimization

subroutine KEEP
peaks select "*,*"
end

noeassign peaks=$peaks prot=$prot keep=$keep autoaco
<pre\>

=== '''A Simple Automated Structure Calculation Using CYANA 3.0''' ===

This section provides an example of a standard automatic NOESY assignment calculation using CYANA 3.0 on a monomeric protein.

==== Input Files ====

Collect the following files in your directory (see the attached files for examples and formatting): 

*[[Media:CYANA30ex_init.cya|init.cya]]:  initialization file. Defines the protein name (i.e., PROT), residue library(ies), number of processors used, and rmsd residue range.
*[[Media:CYANA3ex_CALC.cya|CALC.cya]]:  structure calculation file: Defines the peak lists, tolerances, any NOE calibration parameters (default is automatic calibration), total number of structures calculated in each cycle, number of structures with lowest target function retained after each cycle, number of torsion angle dynamics steps, random seed.
*[[Media:CYANA3ex_PfR193A.seq|PROT.seq]]:  protein sequence file.
*[[Media:CYANA3ex_PfR193A.aco|PROT.aco]]:  dihedral angle constraint file.
*[[Media:CYANA3ex_PfR193A_h2o.prot|filename.prot]]:  chemical shift assignment list.  You should make all degenerate geminal proton assignments Q's, as well as degenerate side chain aromatics (HD1/HD2 and HE1/HE2).
**if your assignments are in a bmrb file (2.1), start cyana, read in the bmrb file, and then write the shifts out to a prot file as follows:
<pre> Open project in cyana 3.0:
read bmrb [finename.bmrb]
write prot [filename.prot]</pre>
*[[Media:CYANA3ex_PfR193A_n15.peaks|filename1.peaks]], [[Media:CYANA3ex_PfR193A_c13_h2o_ali.peaks|filename2.peaks]], [[Media:CYANA3ex_PfR193A_c13_h2o_aro.peaks|filename3.peaks]]:  NOESY peak lists in XEASY format.  The peak lists are unassigned.
*[[Media:CYANA3ex_ssa.cya|ssa.cya]]:  file with stereospecific assignments defined.
*other:  other constraint files such as, manual upper and lower distance limits, hydrogen bond constraints.

==== Running the Program ====

To run CYANA 3.0 on our cluster at CABM, login to master3 and type:
<pre> /farm/software/cyana3.0/bin/cyana CALC > log.out
</pre>
==== Output Files ====

A CYANA structure calculation run will produce .pdb, .upl, .noa and .ovw files for each cycle and the final cycle, as well as log and ramachandran files for the run.

The command <tt>cyanatable</tt> produces a summary table of an automated NOE assignment structure calculation run.

 

== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/9367762?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2 1.    Güntert, P,, Mumenthaler, C. and Wüthrich, K. (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. ''J. Mol. Biol. 273'', 283-298.]

[http://www.ncbi.nlm.nih.gov/pubmed/12051947?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=5 2.    Herrmann, T., Güntert P. and Wüthrich, K. (2002) Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA.  ''J. Mol. Biol. 319'' , 209-227.] 

[http://www.ncbi.nlm.nih.gov/pubmed/2692701?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=9 3.    Neri, D., Szyperski, T., Otting, G., Senn, H. and Wüthrich, K. (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. ''Biochemistry 28'', 7510-7516.]

4.    Momany, F.A., McGuire, R.F., Burgess, A.W. and Scheraga, H.A. (1975)  Energy parameters in polypeptides. VII. Geometric parameters, partial atomic charges, nonbonded interactions, hydrogen bond interactions, and intrinsic torsional potentials for the naturally occurring amino acids.  ''J. Phys. Chem. 79'', 2361-2381.

5.    Nemethy, G., Pottle, M.S. and Scheraga, H.A. (1983)  Energy parameters in polypeptides. 9. Updating of geometrical parameters, nonbonded interactions, and hydrogen bond interactions for the naturally occurring amino acids.  ''J. Phys. Chem. 87'', 1883-1887.

CYANA

2011-10-24T21:41:36Z

Alex: /* Sequence File */

== '''Introduction''' ==

CYANA is a macromolecular structure calculation algorithm based on simulated annealing molecular dynamics calculations in torsional angle space, in contrast to Cartesian space [1,2].  Here the only degrees of freedom are torsion angles with covalent structure parameters kept fixed, thereby significantly decreasing the number of degrees of freedom in the calculation.

The current version of CYANA is 3.0, and it is capable of handling orientational (i.e., RDC) constraints.  There is now a nice [http://www.cyana.org/wiki/index.php/CYANA_3.0_Reference_Manual CYANA 3.0 wiki] with file explanations, tutorials and theory.

In the sections below we describe input files and protocols used in the NESG for both CYANA 2.1 and 3.0.

 

== '''CYANA 2.1''' ==

=== Residue Library ===

CYANA 2.1 uses a new library ~/lib/cyana.lib. It is loaded by the <tt>cyanalib</tt> command. For back-compatibility there is also the old DYANA library dyana.lib (loaded with <tt>dyanalib</tt>, of course).

The main difference is larger van der Waals radii. This will give you a larger target function than DYANA, but also better clash scores.

Residue nomenclature is also different - there are no charged species, like ARG+ or GLU-.

 

=== Atom Nomenclature ===

Atom nomenclature was made compatible with BMRB standard. The deviations from XEASY/DYANA conventions are: HN <-> H, HA1 <-> HA2 and HA2 <-> HA3 for GLY.

There a is macro '''translate.cya''', which is used to convert input to different formats. For example, to read files with DYANA nomenclature, enter <tt>translate dyana</tt>. To switch back to CYANA 2.1 convention type <tt>translate off</tt>.

 

=== Pseudoatom Treatment ===

Pseudoatom handling is switched by setting '''pseudo=x''', where x is 0, 1, 2, or 3.

With '''pseudo=0''', the default setting, coordinate files *.cor and *.pdb do not contain pseudoatoms. They are calculated implicitly on the run.

Setting '''pseudo=1''' restores the old DYANA behavior with explicit pseudoatoms.

Setting '''pseudo=2''' switches to simplified pseudoatom names, such as HB instead of QB, HD1 instead of QD1, and HD instead of QQD of Leu. This is the setting to be used when reading chemical shifts from CARA. Coordinate files will contain explicit pseudoatoms, as with '''pseudo=1'''

Setting '''pseudo=3''' allows X-Plor/CNS pseudoatom names, like HX* instead of QX. For some reason using '''translate xplor''' is not enough to do the conversion for all the atoms.

 

=== The Initialization File:  init.cya ===

The init.cya is a local initialization file, which is read when cyana starts. It should be located in the directory where CYANA is run. In a given project the same file can be used for nearly all calculations.

Create your own init.cya file with the following lines in a text editor or download this template [[Media:CYANA_init.cya|init.cya]] file: 
<pre>name:=XXXX # Replace XXXX with NESG ID
nproc=2 # Number of processors on a workstation
rmsdrange:=20..72 # RMSD reported for these residues after structure calculation
# Read the standard and special libraries
cyanalib
read lib $cyanadir/lib/special.lib append
pseudo=2 # Allows HB, HD, etc. pseudoatom names, use with CARA
read seq $name # Initialize
</pre>
Replace XXXX with your NESG target ID. It is convenient to have the sequence and atomlist files named as <tt>XXXX.seq</tt> and <tt>XXXX.prot</tt>.

*<tt>nproc</tt> defines the number of processors on a workstation.
*<tt>rmsdrange</tt> is only used in structure calculation. Set the range to a valid residue range. From NOE patterns you can exclude flexible N- and C-terminal parts. If you have a flexible loop in the middle you can specify the range as <tt>10..30,40..70</tt>.
*<tt>cyanalib</tt> reads the default <tt>cyana.lib</tt> residue library. The <tt>special.lib</tt> library is appended to use non-standard residues, (i.e., His tautomers).
*<tt>pseudo=2</tt> is only necessary to read atom lists created with CARA, because they have H* for pseudoatom labels. Comment it out, or use <tt>pseudo=0</tt> if you take atom lists from XEASY. 

 

=== The Structure Calculation File:  CALC.cya ===

There are calculation demos for automatic assignment (~/demo/auto) and simple structure calculation (~/demo/manual) runs.

Here is a CALC.cya script for automatic NOE assignment: 
<pre>peaks  := c13.peaks,n15.peaks,aro.peaks # names of NOESY peak lists
prot  := demo # names of chemical shift lists
constraints := demo.aco # additional (non-NOE) constraints
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..100 # residue range for RMSD calculation
randomseed  := 434726 # random number generator seed

noeassign peaks=$peaks prot=$prot autoaco
</pre>
To prevent CYANA from changing existing peak assignments you need to define a subroutine to select the peaks to keep: 
<pre>peaks  := c13.peaks,n15.peaks,aro.peaks # names of NOESY peak lists
prot  := demo # names of chemical shift lists
constraints := demo.aco # additional (non-NOE) constraints
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..100 # residue range for RMSD calculation
randomseed  := 434726 # random number generator seed

subroutine KEEP
peaks select "*, * number=2..7999"
end

noeassign peaks=$peaks prot=$prot autoaco keep=KEEP
</pre>
Here, subroutine <tt>KEEP</tt> is used to keep the assignments for peaks with peak numbers from 2 to 7999.

 

Here is a CALC.cya script for manual structure calculation: 
<pre>peaks  := c13,n15,aro # names of peak lists
prot  := demo # names of proton lists
tolerance  := 0.040,0.030,0.45 # chemical shift tolerances
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)
calibration:= 6.7E5,8.2E5,8.0E4 # calibration constants (will be determined
# automatically, if commented out)
dref  := 4.2 # average upper distance limit for
# automatic calibration

if (master) then

# ---- check consistency of peak and chemical shift lists----

peakcheck peaks=$peaks prot=$prot

# ---- calibration ----

calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref
peaks calibrate "**" simple
write upl $name-in.upl
distance modify
write upl $name.upl

end if
synchronize

# ---- structure calculation ----

read seq $name.seq # re-read sequence to initialize
read upl $name.upl # read upper distance limits
read aco $name.aco # read angle constraints
seed=5671 # random number generator seed
calc_all structures=100 command=anneal steps=10000 # calculate 100 conformers
overview $name.ovw structures=20 pdb # write overview file and coordinates
</pre>
Note the order in which tolerances are given.

The '''calibration''' field can be left empty, in this case '''dref''' will be used to derive calibration constants. If '''dref''' is not specified noeassign.cya will use a default value of 4.0. During calculation noeassign.cya will also relax the calibration if needed (that is in "elastic" mode, which is the default).

'''constraints''' need not be non-NOE despite what the comment says. You can add *.aco, *.upl, *.lol, and even *.cya macros for stereospecific assignments (haven't tested it yet, but that's the way CYANA adds stereospecific assignments in the final round).

'''master''' and '''synchronize''' keywords are needed for running on a cluster.

'''peakcheck''' checks the peaklist assignments against the atom list. Always check CYANA output for '''peakcheck''' results - those huge upl violations may be caused by mis-assigned peaks.

 

=== NOE Calibration ===

CYANA 2.1 by default does not use explicit pseudoatom corrections in distance constraints. Instead, these corrections are applied implicitly on-the-fly. This behavior is turned on by setting '''expand=1'''.

Calibration is thus performed with the undocumented statement '''peaks calibrate "**" simple'''. Trivial calculations show, however, that this command uses a simple r^-6 calibration without adding pseudoatom corrections.

Old calibration macros, such as '''calibrate.cya''' and '''caliba.cya''' are still allowed, but they do add explicit pseudoatom corrections. So if want to use them, don't forget to set '''expand=0'''. Omitting it will result in applying corrections twice, making the corresponding constraints very loose.

This is, of course, a matter of huge confusion since both methods produce otherwise identical *.upl files. Be sure you know HOW you calibrate your NOEs.

To modify upper and lower distance cutoffs for NOE calibration, use '''set upl_values:=2.4,6.0'''. The defaults are 2.4 and 5.5.

For a more detailed description of NOE calibration using CYANA, follow this [[NOE Calibration Using CYANA|link]].

 

=== Dihedral Angle Constraints ===

In CYANA, dihedral angle constraints are specified in a <tt>.aco</tt> file.

Dihedral angle constraints for structure calculation in CYANA can come from a variety of sources.  For example, the [[FOUND|FOUND]] module derives dihedral angle constraints based on local NOE data.

Programs such as [[TALOS|TALOS]] provide backbone phi and psi torsion angle constraints based on chemical shifts.  In our structure determination pipeline we often make use of TALOS-derived backbone torsion angle constraints in our calculations. 

 

=== Stereospecific Assignments ===

Constraints for diastereotopic atoms (such as HB2, HB3) are treated as ambiguous by CYANA. This is switched on with '''swap=1'''.

For the manual run you may want to have '''swap=0''' to be compatible with DYANA behavior. This option is apparently not necessary when distance modification is applied.

Distance modification does not affect Phe and Tyr ring atoms HD1/2 and HE1/2. Therefore, if you have degenerate ring chemical shift (as is almost always the case) make sure you have them labeled QD and QE

External stereospecific assignments determined with [[GLOMSA|'''GLOMSA''']] or with the help of a fractionally (i.e., 5%) 13C-labeled sample [3] can be defined with a custom macro like this: 
<pre> # VAL
atom stereo "QG1 25 36 38 87"
atom stereo "QG1 43 90"
atom swap "QG1 43 90"
# LEU
atom stereo "QD1 60 63 97"
atom stereo "QD1 35 56"
atom swap "QD1 35 56"
</pre>
Here the syntax of CYANA 2.1 requires double quotes. For some strange reason, methyl groups should be written with the letter "Q" even if '''pseudo=2''' is used.

 

=== NOESY Peak Lists ===

CYANA 2.1 can produce multiple assignments for a peak. Below is a part of an aliphatic NOESY peaklist with peak #6 having two assignments. #VC tags specify the weights given to individual assignments. Calibration of this peak yields two constraints splitting the peak integral according to these weights. 
<pre># Number of dimensions 3
#FORMAT xeasy3D
#INAME 1 H
#INAME 2 C
#INAME 3 h
#CYANAFORMAT HCh
1 4.147 51.731 1.474 3 U 7.953E+03 0.000E+00 - 0 2234 2233 2238 #QU 1.000 #SUP 1.00
2 4.147 51.731 4.251 4 U 4.181E+03 0.000E+00 - 0 0 0 0
3 4.147 51.731 7.791 3 U 6.017E+03 0.000E+00 - 0 2234 2233 232 #QU 1.000 #SUP 1.00
4 1.474 22.186 0.515 3 U 1.481E+03 0.000E+00 - 0 2390 2389 1417 #QU 0.981 #SUP 0.98
5 1.474 22.186 1.249 3 U 2.610E+04 0.000E+00 - 0 2390 2389 1706 #QU 0.987 #SUP 0.99
6 1.474 22.186 2.635 3 U 1.396E+03 0.000E+00 - 0 2390 2389 1715 #VC 0.47897 #QU 0.774 #SUP 0.96
2390 2389 1815 #VC 0.52103 #QU 0.813 #SUP 0.96
7 1.474 22.186 3.863 3 U 1.418E+04 0.000E+00 - 0 2390 2389 1657 #QU 0.885 #SUP 0.88
8 1.474 22.186 4.147 3 U 1.448E+04 0.000E+00 - 0 2238 2237 2234 #QU 1.000 #SUP 1.00
</pre>
The peaklists produced by CYANA 2.1 are not backwards-compatible with XEASY, but there are Lua scripts, which can read them into CARA including the information on ambiguous assignments. UBNMR should also be able to handle them in the future.

When supplying completely unassigned peaks for automatic NOE assignment it is necessary to include a line like '''#CYANAFORMAT HCh''' in the header.

 

== '''CYANA 3.0''' ==

Again please consult the [http://www.cyana.org/wiki/index.php/CYANA_3.0_Reference_Manual CYANA 3.0 wiki] for complete details on file formats, input files for CYANA, and other documentation.

=== Residue Library ===

A [http://www.cyana.org/wiki/index.php/Residue_library_file residue library] defines all properties of a residue including atom types, the nomenclature, the dihedral angle definitions, the covalent connectivities and the standard geometry. The standard geometry of the ECEPP/2 force field [4,5] is used for all amino acid residue types.  Standard residues are collected in the '''cyana.lib''' library; special residue types are in the '''special.lib''' library.

=== Sequence File ===

The [http://www.cyana.org/wiki/index.php/Sequence_file sequence file] (.seq) defines the sequence of the molecule you are working with.  Special residue types (i.e., oxdized cysteine, histidine tautomers, and cis-peptide bonds) can also be defined in the sequence file as follows:

*oxidized cysteine:  CISS
*charged histidine:  HIS+
*Nε2H neutral tautomer:  HIST (the default HIS specifies the Nδ1H neutral tautomer).
*cis-peptide bond:  place a "c" before the residue name;  i.e., cPRO
*invisible intermolecular linkers:  PL, LL, LL2, LL5, LP

 

=== Automated Structure calculation ===

<pre>
peaks := n,ali,aro # names of NOESY peak lists
prot := $name # names of chemical shift lists
restraints := talos.aco,stereo.cya # additional (non-NOE) constraints
tolerance := 0.04,0.02,0.4 # chemical shift tolerances
# order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a)
upl_values := 2.4,5.5 # calibration cutoffs
cut_upl=0.05
calibration_constant:= # NOE calibration parameters
structures := 100,20 # number of initial, final structures
steps := 10000 # number of torsion angle dynamics steps
rmsdrange := 20..102 # residue range for RMSD calculation
randomseed := 562 # random number generator seed
calibration_dref := 4.0 # average distance for calibration, default 4.0
keep := # set to KEEP to retain existing assignments

weight_rdc = 0.002 # weight for RDC restraints
cut_rdc = 1.0 # cut-off for RDC violations
opt_tensor = 1 # alignment tensor optimization

subroutine KEEP
peaks select "*,*"
end

noeassign peaks=$peaks prot=$prot keep=$keep autoaco
<pre/>

=== '''A Simple Automated Structure Calculation Using CYANA 3.0''' ===

This section provides an example of a standard automatic NOESY assignment calculation using CYANA 3.0 on a monomeric protein.

==== Input Files ====

Collect the following files in your directory (see the attached files for examples and formatting): 

*[[Media:CYANA30ex_init.cya|init.cya]]:  initialization file. Defines the protein name (i.e., PROT), residue library(ies), number of processors used, and rmsd residue range.
*[[Media:CYANA3ex_CALC.cya|CALC.cya]]:  structure calculation file: Defines the peak lists, tolerances, any NOE calibration parameters (default is automatic calibration), total number of structures calculated in each cycle, number of structures with lowest target function retained after each cycle, number of torsion angle dynamics steps, random seed.
*[[Media:CYANA3ex_PfR193A.seq|PROT.seq]]:  protein sequence file.
*[[Media:CYANA3ex_PfR193A.aco|PROT.aco]]:  dihedral angle constraint file.
*[[Media:CYANA3ex_PfR193A_h2o.prot|filename.prot]]:  chemical shift assignment list.  You should make all degenerate geminal proton assignments Q's, as well as degenerate side chain aromatics (HD1/HD2 and HE1/HE2).
**if your assignments are in a bmrb file (2.1), start cyana, read in the bmrb file, and then write the shifts out to a prot file as follows:
<pre> Open project in cyana 3.0:
read bmrb [finename.bmrb]
write prot [filename.prot]</pre>
*[[Media:CYANA3ex_PfR193A_n15.peaks|filename1.peaks]], [[Media:CYANA3ex_PfR193A_c13_h2o_ali.peaks|filename2.peaks]], [[Media:CYANA3ex_PfR193A_c13_h2o_aro.peaks|filename3.peaks]]:  NOESY peak lists in XEASY format.  The peak lists are unassigned.
*[[Media:CYANA3ex_ssa.cya|ssa.cya]]:  file with stereospecific assignments defined.
*other:  other constraint files such as, manual upper and lower distance limits, hydrogen bond constraints.

==== Running the Program ====

To run CYANA 3.0 on our cluster at CABM, login to master3 and type:
<pre> /farm/software/cyana3.0/bin/cyana CALC > log.out
</pre>
==== Output Files ====

A CYANA structure calculation run will produce .pdb, .upl, .noa and .ovw files for each cycle and the final cycle, as well as log and ramachandran files for the run.

The command <tt>cyanatable</tt> produces a summary table of an automated NOE assignment structure calculation run.

 

== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/9367762?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2 1.    Güntert, P,, Mumenthaler, C. and Wüthrich, K. (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. ''J. Mol. Biol. 273'', 283-298.]

[http://www.ncbi.nlm.nih.gov/pubmed/12051947?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=5 2.    Herrmann, T., Güntert P. and Wüthrich, K. (2002) Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA.  ''J. Mol. Biol. 319'' , 209-227.] 

[http://www.ncbi.nlm.nih.gov/pubmed/2692701?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=9 3.    Neri, D., Szyperski, T., Otting, G., Senn, H. and Wüthrich, K. (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. ''Biochemistry 28'', 7510-7516.]

4.    Momany, F.A., McGuire, R.F., Burgess, A.W. and Scheraga, H.A. (1975)  Energy parameters in polypeptides. VII. Geometric parameters, partial atomic charges, nonbonded interactions, hydrogen bond interactions, and intrinsic torsional potentials for the naturally occurring amino acids.  ''J. Phys. Chem. 79'', 2361-2381.

5.    Nemethy, G., Pottle, M.S. and Scheraga, H.A. (1983)  Energy parameters in polypeptides. 9. Updating of geometrical parameters, nonbonded interactions, and hydrogen bond interactions for the naturally occurring amino acids.  ''J. Phys. Chem. 87'', 1883-1887.

Creating NOESY peaklists with CARA

2011-10-18T19:35:21Z

Alex:

One can start either by creating an empty peaklist, or by exporting inferred peak and spinliks from PolyScope. After that it is possible to add peaks interactively or with a script. The fastest method seems to be picking peaks automaticaly with the 'PickAll3DNOESY' script from an empty peaklist.

== '''Simulate short and sequential peaks''' ==

# Run 'ExportToCSI' script to generate an input file for CSI
# Run the CSI program
# Run the 'CreateShortSequentialSpinLinks' script to generate spin-links based on CSI prediction
# Remove all unnecessary spins, such as draft assignments (e.g. '?HA'), projected spins (e.g. 'HA-1') and GFT pseudospins (e.g. 'CApCB'). Bulk remove is possible with scripts such as RemoveSins, RemoveProjectedSpins and RemoveGFTSpins.
# Open an HSQC spectrum in PolyScope window with the corresponding NOESY spectrum loaded in the strip. In the 'Strip' menu make sure that the 'Show Spin Links' box is checked, and the 'Show Inferred Peaks' is unchecked. Select 'File' -> 'Export...' -> 'Strip Peaks to MonoScope...' to create an initial peaklist with existing assignments.

* Note that certain spinlinks (such as HE <-> N in Lys, for example) will yield undetectable peaks. Therefore, integrate the peak list first and then use the 'PeakListReport' script to delete all peaks weaker than a certain threshold.
* Some peaks may have significant overlap, or have contributions from yet unassigned long-range peaks. Interactive refinement is necessary.

== '''Automated peak picking''' ==

# For each peaklist define the typical peak linewidths using '''Intergrate -> Tune Peak Model...''' in MonoScope.
# Set 'DeafultPeaklist' and 'PeakListToSpectrumMapping' attributes of the corresponding spectrum according to the peaklist.
# Run the 'PickAll3DNOESY' script to automatically pick peaks.
# Run the 'PeakListReport' script to delete diagonal peaks.
# Refine the resulting peaklist by interactivelly scanning all strips.

Structure Calculation Using CS-Rosetta

2011-10-18T16:55:50Z

Alex:

== '''Introduction''' ==

The CS-ROSETTA approach [1,2] combines the Monte Carlo based structure assembly program ROSETTA with empirical structural information obtained from backbone and 13Cβ chemical shift data.  The robust CS-ROSETTA protocol is capable of successfully predicting 3D protein structures up to 15 kDa in size [1].  A complete description of the program along with downloads are available from the Bax laboratory web site:

http://spin.niddk.nih.gov/bax/software/CSROSETTA/index.html

 

== '''CS-ROSETTA Servers''' ==

* http://condor.bmrb.wisc.edu/bbee/rosetta/ Server at BMRB

== '''CS-ROSETTA Protocol at UB''' ==

=== '''Random Coil Index Prediction''' ===

Perform flexible region prediction on the [http://wishart.biology.ualberta.ca/rci/cgi-bin/rci_cgi_1_e.py RCI Web Page].

RCI will take a bmrb file in the old format, as produced by CYANA 1.0 (the new BMRB format has an extra "chain" column). Unlike AutoStructure input file, the sequence field should be left in place.

Use an init.cya file: 
<pre> name:=XXXX # Replace XXXX with NESG ID
cyanalib # Read the standard library
pseudo=2 # Allows HB, HD, etc. pseudoatom names, use with CARA
read seq $name # Initialize</pre>
If you are using proton list from CARA, convert it first to "dyana" format with Cyana 2.1: 
<pre> read prot XXXX.prot
pseudo=0
translate dyana
write prot XXXX_dyana</pre>
Use cyana 1.0.5 to prepare a bmrb file:
<pre> read prot XXXX_dyana.prot
bmrblist XXXX.bmrb
</pre>
Make sure you change the <tt>_Chem_shift_ambiguity_type</tt> tag to <tt>_Chem_shift_ambiguity_code</tt>; RCI will report an error if you don't do it.

Flexible N- and C-terminal tails should be removed for CS-ROSETTA calculation to reduce CPU time. Flexible loop regions will later be excluded from calculation of all-atom energy.

=== '''Generating MFR fragments on U2 cluster at SUNY Buffalo''' ===

Copy the <tt>runCSRjob.com</tt> file into the working directory and change the number of fragments to be generated.

Type <tt>qsub runCSRjob.pbs</tt> to submit the job into queue. This calculation takes ~2 hours for 1000 fragments for a small protein, therefore it cannot be run on a master node.

=== '''Running CS-Rosetta on U2 cluster at SUNY Buffalo''' ===

Go to the <tt>rosetta</tt> subdirectory. Figure out how many parallel Rosetta jobs you will need to run. Things to consider are:

*The total number of fragments to be calculated
*The maximum wall-time for a single job is 72 h
*It takes ~10 min to calculate a single structure of a small protein on a single CPU

 Type <tt>./runRosetta.csh N</tt>, where =N= is the number of parallel Rosetta jobs

 

'''CS-ROSETTA Protocol at CABM'''

It is assumed that cs-rosetta2.3.0, rosetta2.3.0, NMRPipe-2008 are already installed and running in your cluster (see the [http://spin.niddk.nih.gov/bax/software/CSROSETTA/index.html Bax laboratory web site] for instructions).  In addition, the the following activation commands may need to be issued in your local shell: 

*For c-shell (csh, tcsh)
<pre>source /farm/software/NMRPipe-2008/com/nmrInit.linux9.com

source /farm/software/cs-rosetta2.3.0/com/csrosettaInit.com</pre>
*For bash shell (sh, bash)
<pre>source /farm/software/NMRPipe-2008/com/nmrInit.linux9.sh

source /farm/software/cs-rosetta2.3.0/com/csrosettaInit.sh
</pre>
=== Protocol for running CS-ROSETTA at CABM ===

Start from a chemical shift file in bmrb 2.1 format including the complete header.    Here is an example bmrb file in the correct format, the scripts are rather unforgiving of format inconsistencies. 

Chemical Shift rosetta uses TALOS format for the chemical shifts so first one needs to convert those into the right format. The right order of actions would be (the software mentioned is available through NMRPipe2008 and CS-rosetta and it will be accessible if the proper intialization was done –see preceeding paragraph). 
<pre>bmrb2talos.com BMRB_cs_file > prot_CS

runCSRjob.com prot_CS</pre>
this is really time-consuming (somewhere between 1 and 4 hours in master2) it will produce a directory called 'rosetta' and under it you will have 

■ aat000_03_05.200_v1_3 ■ aat000_09_05.200_v1_3 ■ paths.txt ■ runRosetta.com ■ t000_.fasta 

the last step will be to run the runRosetta script which contains the Rosetta run instruction code:
<pre>runRosetta.com
</pre>
this runs a single cpu job. In order to make use of the cluster a launching template called lzRosetta was created that sends the calculations over a computer cluster.  The command:
<pre>qsub lzRosetta
</pre>
submits the job.  Depending on the cluster usage, several instances of the above command can be launched to occupy as many cpu as possible.  Rosetta handles the output bookkeeping and increments the decoy counter automatically so that the chosen number of decoys are calculated by the available CPUs.  The number of decoys can be adjusted in the runRosetta script (e.g. -nstruct 1000). 

 

== '''Files for Download''' ==

[[Media:013008_ref_caps.bmrb|Input.bmrb]]:  Bmrb file in 2.1 format.  Input for bmrb2talos.com command.  Note the formatting.

[[Media:RrR43_CS.txt|Output_CS]]:  Chemical shift file produced by bmrb2talos.com command.

[[Media:LzRosetta.txt|lzRosetta]]:  Script for sending CS-Rosetta calculations to a cluster.

== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/18326625?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2 1.  Shen, Y., Lange, O., Delaglio, F., Rossi, P., Aramini, J.M., Liu, G., Eletsky, A., Wu, Y., Singarapu, K.K., Lamak, A., Ignatchenko, A., Arrowsmith, C.H., Szyerpski, T., Montelione, G.T., Baker, D and Bax, A. (2008) Consistent blind protein structure generation from NMR chemical shift data. ''Proc. Natl Acad Sci. 105'', 4585-4590.]

[http://www.ncbi.nlm.nih.gov/pubmed/19034676?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2 2.  Shen, Y., Vernon, R., Baker, D. and Bax, A. (2009) De novo protein structure determination from incomplete chemical shift assignments.  J''. Biomol. NMR 43'', 63-78.]

[http://www.ncbi.nlm.nih.gov/pubmed/20133520?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2 2.  Raman S, Lange OF, Rossi P, Tyka M, Wang X, Aramini J, Liu G, Ramelot TA, Eletsky A, Szyperski T, Kennedy MA, Prestegard J, Montelione GT, Baker D. (2009) NMR structure determination for larger proteins using backbone-only data.  Science''. 327'', 1014-8.]

TALOS

2011-10-18T16:50:56Z

Alex: /* References */

== '''Introduction''' ==

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 <ref><pubmed>10212987</pubmed></ref>. In 2009, the Bax laboratory released a new and improved version of the program called TALOS+<ref><pubmed>19548092</pubmed></ref>. 

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.  For installation questions and other support, you can also e-mail [mailto:shenyang@niddk.nih.gov Yang Shen].

== '''Generating TALOS dihedral angle constraints with CYANA (UB)''' ==

#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.
#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.
#Start CYANA and type:
#:<pre>
#:read prot XXXX.prot
#:taloslist XXXX
#:</pre>
#This will create the TALOS input file <tt>XXXX.tab</tt>. In this file rename all "H" atoms to "HN".
#In a UNIX shell run
#:<pre>
#:talos+ -in XXXX.tab
#:</pre>This will create a file <tt>pred.tab</tt>, which includes an initial summary of the prediction results.
#In a UNIX shell run
#:<pre>
#:rama+ -in XXXX.tab
#:</pre>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>.
#Start CYANA and type:
#:<pre>
#:talosaco pred #or "talos.tab" -- use the appropriate filename
#:write aco talos.aco
#:</pre>

=== '''talosaco.cya macro''' ===

The <tt>talosaco</tt> macro is invoked as:
<pre>talosaco file [factor [width]]</pre>
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>.

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.

See also the <tt>~/demo/details/TalosAngleRestraints.cya</tt> example script in your local CYANA 2.1 installation.

 

=== '''Interactive Refinement of TALOS Predictions''' ===

Guidelines for refining the TALOS output:

*Classify prediction as <tt>Good</tt> only if
**All 10 best database matches fall in a "consistent" region of the Ramachandran map
**Or 9 out of 10 best database matches fall in a consistent region with <tt>phi < 0</tt>, and the one outlier also lies in <tt>phi < 0</tt> half of the map
**Or 9 out of 10 of the best database matches fall in a consistent region with <tt>phi > 0</tt>
*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).

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.

During structure refinement you can refine TALOS predictions against a preliminary structure.
<pre>vina.tcl -in XXXX.tab -ref XXXX.pdb -auto</pre>
and
<pre>rama.tcl -in XXXX.tab -ref XXXX.pdb</pre>
 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).

 

{| border="1"
|-
| Element
| PHI
| PSI
|-
| α-helix
| -60
| -45
|-
| β-sheet
| -140
| 135
|}

 

== '''Using TALOS and TALOS+ at CABM''' ==

=== Preparing for a TALOS+ run ===

*Make a sub-directory in your project for TALOS.
*you will need the following files in your directory:
*a bmrb file in 2.1 format.  Here is an [[Media:PfR193A_062509_2.1f_4CYANA.bmrb|example]].
*[[Media:BMRBParsing.pm|BMRBParsing.pm]]:  BMRB parser
*[[Media:Tab4Talos.txt|Tab4Talos.pl]]:  perl script to prepare input file for TALOS
*[[Media:Talos2dyana_taloserrors.txt|talos2dyana_taloserrors.pl]]:  perl script to prepare a CYANA .aco file
*Run the following command:
<pre> Tab4Talos.pl [.bmrbf] [input4Talos]

</pre>
This make an input chemical shift list for TALOS.  Here is an [[Media:PfR193A_4Talos.input|example]]. 

=== Running TALOS+ and making a dihedral angle constraint file ===

*Next run talos+:
<pre> talos+ -in [input4Talos</pre>
This makes a number of output files including the pred.tab.  

*Next, edit the pred.tab and comment out (#) any lines that do not have the "10 Good" comment. 
*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.  They user can modify this script to make his/her own error limits (i.e., +/- 20 or 30).
<pre> perl talos2dyana_taloserrors.pl pred.tab [output.aco]
</pre>
 

== '''References''' ==

<references />

TALOS

2011-10-18T16:50:23Z

Alex: /* Introduction */

== '''Introduction''' ==

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 <ref><pubmed>10212987</pubmed></ref>. In 2009, the Bax laboratory released a new and improved version of the program called TALOS+<ref><pubmed>19548092</pubmed></ref>. 

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.  For installation questions and other support, you can also e-mail [mailto:shenyang@niddk.nih.gov Yang Shen].

== '''Generating TALOS dihedral angle constraints with CYANA (UB)''' ==

#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.
#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.
#Start CYANA and type:
#:<pre>
#:read prot XXXX.prot
#:taloslist XXXX
#:</pre>
#This will create the TALOS input file <tt>XXXX.tab</tt>. In this file rename all "H" atoms to "HN".
#In a UNIX shell run
#:<pre>
#:talos+ -in XXXX.tab
#:</pre>This will create a file <tt>pred.tab</tt>, which includes an initial summary of the prediction results.
#In a UNIX shell run
#:<pre>
#:rama+ -in XXXX.tab
#:</pre>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>.
#Start CYANA and type:
#:<pre>
#:talosaco pred #or "talos.tab" -- use the appropriate filename
#:write aco talos.aco
#:</pre>

=== '''talosaco.cya macro''' ===

The <tt>talosaco</tt> macro is invoked as:
<pre>talosaco file [factor [width]]</pre>
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>.

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.

See also the <tt>~/demo/details/TalosAngleRestraints.cya</tt> example script in your local CYANA 2.1 installation.

 

=== '''Interactive Refinement of TALOS Predictions''' ===

Guidelines for refining the TALOS output:

*Classify prediction as <tt>Good</tt> only if
**All 10 best database matches fall in a "consistent" region of the Ramachandran map
**Or 9 out of 10 best database matches fall in a consistent region with <tt>phi < 0</tt>, and the one outlier also lies in <tt>phi < 0</tt> half of the map
**Or 9 out of 10 of the best database matches fall in a consistent region with <tt>phi > 0</tt>
*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).

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.

During structure refinement you can refine TALOS predictions against a preliminary structure.
<pre>vina.tcl -in XXXX.tab -ref XXXX.pdb -auto</pre>
and
<pre>rama.tcl -in XXXX.tab -ref XXXX.pdb</pre>
 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).

 

{| border="1"
|-
| Element
| PHI
| PSI
|-
| α-helix
| -60
| -45
|-
| β-sheet
| -140
| 135
|}

 

== '''Using TALOS and TALOS+ at CABM''' ==

=== Preparing for a TALOS+ run ===

*Make a sub-directory in your project for TALOS.
*you will need the following files in your directory:
*a bmrb file in 2.1 format.  Here is an [[Media:PfR193A_062509_2.1f_4CYANA.bmrb|example]].
*[[Media:BMRBParsing.pm|BMRBParsing.pm]]:  BMRB parser
*[[Media:Tab4Talos.txt|Tab4Talos.pl]]:  perl script to prepare input file for TALOS
*[[Media:Talos2dyana_taloserrors.txt|talos2dyana_taloserrors.pl]]:  perl script to prepare a CYANA .aco file
*Run the following command:
<pre> Tab4Talos.pl [.bmrbf] [input4Talos]

</pre>
This make an input chemical shift list for TALOS.  Here is an [[Media:PfR193A_4Talos.input|example]]. 

=== Running TALOS+ and making a dihedral angle constraint file ===

*Next run talos+:
<pre> talos+ -in [input4Talos</pre>
This makes a number of output files including the pred.tab.  

*Next, edit the pred.tab and comment out (#) any lines that do not have the "10 Good" comment. 
*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.  They user can modify this script to make his/her own error limits (i.e., +/- 20 or 30).
<pre> perl talos2dyana_taloserrors.pl pred.tab [output.aco]
</pre>
 

== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/10212987?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=3 1.    Cornilescu, G., Delaglio, F. and Bax, A. (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology. ''J. Biomol. NMR 13'', 289-302.]

[http://www.ncbi.nlm.nih.gov/pubmed/19548092?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1 2.    Shen, Y., Delaglio, F., Cornilescu, G. and Bax, A. (2009) TALOS+: A hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. ''J. Biomol. NMR 44'', 213-223]

TALOS

2011-10-18T16:47:35Z

Alex: /* Using TALOS with CYANA */

== '''Introduction''' ==

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].  In 2009, the Bax laboratory released a new and improved version of the program called TALOS+ [2]. 

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.  For installation questions and other support, you can also e-mail [mailto:shenyang@niddk.nih.gov Yang Shen].

== '''Generating TALOS dihedral angle constraints with CYANA (UB)''' ==

#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.
#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.
#Start CYANA and type:
#:<pre>
#:read prot XXXX.prot
#:taloslist XXXX
#:</pre>
#This will create the TALOS input file <tt>XXXX.tab</tt>. In this file rename all "H" atoms to "HN".
#In a UNIX shell run
#:<pre>
#:talos+ -in XXXX.tab
#:</pre>This will create a file <tt>pred.tab</tt>, which includes an initial summary of the prediction results.
#In a UNIX shell run
#:<pre>
#:rama+ -in XXXX.tab
#:</pre>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>.
#Start CYANA and type:
#:<pre>
#:talosaco pred #or "talos.tab" -- use the appropriate filename
#:write aco talos.aco
#:</pre>

=== '''talosaco.cya macro''' ===

The <tt>talosaco</tt> macro is invoked as:
<pre>talosaco file [factor [width]]</pre>
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>.

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.

See also the <tt>~/demo/details/TalosAngleRestraints.cya</tt> example script in your local CYANA 2.1 installation.

 

=== '''Interactive Refinement of TALOS Predictions''' ===

Guidelines for refining the TALOS output:

*Classify prediction as <tt>Good</tt> only if
**All 10 best database matches fall in a "consistent" region of the Ramachandran map
**Or 9 out of 10 best database matches fall in a consistent region with <tt>phi < 0</tt>, and the one outlier also lies in <tt>phi < 0</tt> half of the map
**Or 9 out of 10 of the best database matches fall in a consistent region with <tt>phi > 0</tt>
*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).

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.

During structure refinement you can refine TALOS predictions against a preliminary structure.
<pre>vina.tcl -in XXXX.tab -ref XXXX.pdb -auto</pre>
and
<pre>rama.tcl -in XXXX.tab -ref XXXX.pdb</pre>
 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).

 

{| border="1"
|-
| Element
| PHI
| PSI
|-
| α-helix
| -60
| -45
|-
| β-sheet
| -140
| 135
|}

 

== '''Using TALOS and TALOS+ at CABM''' ==

=== Preparing for a TALOS+ run ===

*Make a sub-directory in your project for TALOS.
*you will need the following files in your directory:
*a bmrb file in 2.1 format.  Here is an [[Media:PfR193A_062509_2.1f_4CYANA.bmrb|example]].
*[[Media:BMRBParsing.pm|BMRBParsing.pm]]:  BMRB parser
*[[Media:Tab4Talos.txt|Tab4Talos.pl]]:  perl script to prepare input file for TALOS
*[[Media:Talos2dyana_taloserrors.txt|talos2dyana_taloserrors.pl]]:  perl script to prepare a CYANA .aco file
*Run the following command:
<pre> Tab4Talos.pl [.bmrbf] [input4Talos]

</pre>
This make an input chemical shift list for TALOS.  Here is an [[Media:PfR193A_4Talos.input|example]]. 

=== Running TALOS+ and making a dihedral angle constraint file ===

*Next run talos+:
<pre> talos+ -in [input4Talos</pre>
This makes a number of output files including the pred.tab.  

*Next, edit the pred.tab and comment out (#) any lines that do not have the "10 Good" comment. 
*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.  They user can modify this script to make his/her own error limits (i.e., +/- 20 or 30).
<pre> perl talos2dyana_taloserrors.pl pred.tab [output.aco]
</pre>
 

== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/10212987?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=3 1.    Cornilescu, G., Delaglio, F. and Bax, A. (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology. ''J. Biomol. NMR 13'', 289-302.]

[http://www.ncbi.nlm.nih.gov/pubmed/19548092?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1 2.    Shen, Y., Delaglio, F., Cornilescu, G. and Bax, A. (2009) TALOS+: A hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. ''J. Biomol. NMR 44'', 213-223]

NMR determined Rotational correlation time

2011-10-13T22:43:05Z

Alex:

[[Image:T1wiki fig5.png|right|400px]] Brownian rotation diffusion of a particle in solution has a characteristic time constant called rotational correlation time (τc). 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

:<math>\tau_c=\frac{4\pi\eta r^3}{3kT}</math>,

where η is the viscosity of the solvent, ''r'' is the effective hydrodynamic radius of the protein molecule, ''k'' is the Boltzmann constant and ''T'' is the temperature. The hydrodynamic radius can be estimated from the molecular weight of the protein (M) as

:<math>r\approx\sqrt[3]{\frac{3M}{4\pi\rho N_a}}+r_w</math>,

where ρ is the average density for proteins (1.37 g/cm3), ''N''''a'' is the Avogadro's number and ''r''''w'' 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 τc of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kDa.

 

For rigid protein molecules, in the limit of slow molecular motion (τc >> 0.5 ns) and high magnetic field (500 MHz or greater), a closed-form solution for τc as a function of the ratio of the longitudinal (T1) and transverse (T2) 15N relaxation times exists:

:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,

where νN is the 15N resonance frequency (in Hz). This equation is derived from Eq. 8 in ref.<ref><pubmed>2690953</pubmed></ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.

 Average 15N T1 and T2 relaxation times for a given protein can be measured using 1D 15N-edited relaxation experiments <ref><pubmed>7514039</pubmed></ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide 1H 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 τc vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T1/T2 method is suitable for proteins with molecular weight of up to MW ≈ 25 kDa. Accurate measurement of the diminishing 15N T2 becomes difficult for larger proteins and cross-correlated relaxation rates are measured instead.

 

Below is a table of rotational correlation time values compiled for known monomeric NESG targets (J. Aramini; June 2010).  All data was recorded on a Bruker 600 NMR instrument at 298 K.  The molecular weight for each target takes into account isotopic enrichment and the presence of affinity purification tags (if any).

 

{| cellspacing="1" cellpadding="1" border="1" align="center" style="width: 1047px; height: 393px;"
|-
| width="250" align="center" | '''NESG target (isotope labeling)'''
| align="center" | '''MW (kDa)'''
| align="center" | '''15N ''T''1 (ms)'''
| align="center" | '''15N ''T''2 (ms)'''
| align="center" | '''τc (ns)'''
|-
| width="250" align="center" | PsR76A  (NC5)
| align="center" | 7.2
| align="center" | 478.0
| align="center" | 128.0
| align="center" | 5.10
|-
| width="250" align="center" | VfR117 (NC)
| align="center" | 11.2
| align="center" | 605.0
| align="center" | 119.0
| align="center" | 6.30
|-
| width="250" align="center" | SyR11 (NC5)
| align="center" | 12.4
| align="center" | 630.0
| align="center" | 104.0
| align="center" | 7.10
|-
| width="250" align="center" | ER541-37-162 (NC5)
| align="center" | 15.8
| align="center" | 729.0
| align="center" | 66.5
| align="center" | 10.0
|-
| width="250" align="center" | ER540 (NC5)
| align="center" | 18.8
| align="center" | 909.0
| align="center" | 66.5
| align="center" | 11.3
|-
| width="250" align="center" | SoR190 (NC)
| align="center" | 13.8
| align="center" | 697.5
| align="center" | 100.9
| align="center" | 7.70
|-
| width="250" align="center" | TR80 (NC5)
| align="center" | 10.5
| align="center" | 612.8
| align="center" | 102.9
| align="center" | 7.00
|-
| width="250" align="center" | Ubiquitin (NC)
| align="center" | 9.0
| align="center" | 441.8
| align="center" | 144.6
| align="center" | 4.40
|-
| width="250" align="center" | HR2873B (NC)
| align="center" | 10.7
| align="center" | 492.0
| align="center" | 115.0
| align="center" | 5.70
|-
| width="250" align="center" | B-domain (NC)
| align="center" | 7.2
| align="center" | 423.5
| align="center" | 153.3
| align="center" | 4.05
|-
| width="250" align="center" | BcR97A (NC)
| align="center" | 13.1
| align="center" | 705.8
| align="center" | 80.6
| align="center" | 8.80
|-
| width="250" align="center" | PfR193A (NC)
| align="center" | 13.6
| align="center" | 733.9
| align="center" | 80.9
| align="center" | 9.00
|-
| width="250" align="center" | MvR76 (NC)
| align="center" | 20.2
| align="center" | 1015.0
| align="center" | 64.5
| align="center" | 12.2
|-
| width="250" align="center" | DvR115G (NC)
| align="center" | 10.9
| align="center" | 608.7
| align="center" | 115.6
| align="center" | 6.50
|-
| width="250" align="center" | MrR110B (NC5)
| align="center" | 11.8
| align="center" | 707.0
| align="center" | 99.2
| align="center" | 7.80
|-
| width="250" align="center" | VpR247 (NC5)
| align="center" | 12.5
| align="center" | 661.2
| align="center" | 88.3
| align="center" | 8.05
|-
| width="250" align="center" | BcR147A (NC)
| align="center" | 11.9
| align="center" | 645.0
| align="center" | 104.0
| align="center" | 7.20
|-
| width="250" align="center" | WR73 (NC5)
| align="center" | 21.9
| align="center" | 1261.0 *
| align="center" | 41.3 *
| align="center" | 13.0
|-
| width="250" align="center" | NsR431C (NC5)
| align="center" | 16.8
| align="center" | 855.5
| align="center" | 71.2
| align="center" | 10.6
|-
| width="250" align="center" | StR82 (NC)
| align="center" | 9.2
| align="center" | 537.3
| align="center" | 100.4
| align="center" | 6.6
|}

*data obtained on an 800 MHz Bruker spectrometer at 298 K.

 

== '''Protocols for Bruker and Varian NMR Instruments''' ==

*[[Measuring 15N T1 and T2 Relaxation Times on Bruker Instruments|Bruker protocol]]
*[[Estimation of rotational correlation time|Varian protocol]]

 

== '''References''' ==

<references />

NMR determined Rotational correlation time

2011-10-13T22:42:23Z

Alex:

[[Image:T1wiki fig5.png|right|400px]] Brownian rotation diffusion of a particle in solution has a characteristic time constant called rotational correlation time (τc). 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

:<math>\tau_c=\frac{4\pi\eta r^3}{3kT}</math>,

where η is the viscosity of the solvent, ''r'' is the effective hydrodynamic radius of the protein molecule, ''k'' is the Boltzmann constant and ''T'' is the temperature. The hydrodynamic radius can be estimated from the molecular weight of the protein (M) as

:<math>r\approx\sqrt[3]{\frac{3M}{4\pi\rho N_a}}+r_w</math>,

where ρ is the average density for proteins (1.37 g/cm3), ''N''''a'' is the Avogadro's number and ''r''''w'' 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 τc of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kDa.

 

For rigid protein molecules, in the limit of slow molecular motion (τc >> 0.5 ns) and high magnetic field (500 MHz or greater), a closed-form solution for τc as a function of the ratio of the longitudinal (T1) and transverse (T2) 15N relaxation times exists:

:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,

where νN is the 15N resonance frequency (in Hz). This equation is derived from Eq. 8 in ref.<ref><pubmed>2690953</pubmed><doi>10.1021/bi00449a003</doi>
</ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.

 Average 15N T1 and T2 relaxation times for a given protein can be measured using 1D 15N-edited relaxation experiments <ref><pubmed>7514039</pubmed></ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide 1H 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 τc vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T1/T2 method is suitable for proteins with molecular weight of up to MW ≈ 25 kDa. Accurate measurement of the diminishing 15N T2 becomes difficult for larger proteins and cross-correlated relaxation rates are measured instead.

 

Below is a table of rotational correlation time values compiled for known monomeric NESG targets (J. Aramini; June 2010).  All data was recorded on a Bruker 600 NMR instrument at 298 K.  The molecular weight for each target takes into account isotopic enrichment and the presence of affinity purification tags (if any).

 

{| cellspacing="1" cellpadding="1" border="1" align="center" style="width: 1047px; height: 393px;"
|-
| width="250" align="center" | '''NESG target (isotope labeling)'''
| align="center" | '''MW (kDa)'''
| align="center" | '''15N ''T''1 (ms)'''
| align="center" | '''15N ''T''2 (ms)'''
| align="center" | '''τc (ns)'''
|-
| width="250" align="center" | PsR76A  (NC5)
| align="center" | 7.2
| align="center" | 478.0
| align="center" | 128.0
| align="center" | 5.10
|-
| width="250" align="center" | VfR117 (NC)
| align="center" | 11.2
| align="center" | 605.0
| align="center" | 119.0
| align="center" | 6.30
|-
| width="250" align="center" | SyR11 (NC5)
| align="center" | 12.4
| align="center" | 630.0
| align="center" | 104.0
| align="center" | 7.10
|-
| width="250" align="center" | ER541-37-162 (NC5)
| align="center" | 15.8
| align="center" | 729.0
| align="center" | 66.5
| align="center" | 10.0
|-
| width="250" align="center" | ER540 (NC5)
| align="center" | 18.8
| align="center" | 909.0
| align="center" | 66.5
| align="center" | 11.3
|-
| width="250" align="center" | SoR190 (NC)
| align="center" | 13.8
| align="center" | 697.5
| align="center" | 100.9
| align="center" | 7.70
|-
| width="250" align="center" | TR80 (NC5)
| align="center" | 10.5
| align="center" | 612.8
| align="center" | 102.9
| align="center" | 7.00
|-
| width="250" align="center" | Ubiquitin (NC)
| align="center" | 9.0
| align="center" | 441.8
| align="center" | 144.6
| align="center" | 4.40
|-
| width="250" align="center" | HR2873B (NC)
| align="center" | 10.7
| align="center" | 492.0
| align="center" | 115.0
| align="center" | 5.70
|-
| width="250" align="center" | B-domain (NC)
| align="center" | 7.2
| align="center" | 423.5
| align="center" | 153.3
| align="center" | 4.05
|-
| width="250" align="center" | BcR97A (NC)
| align="center" | 13.1
| align="center" | 705.8
| align="center" | 80.6
| align="center" | 8.80
|-
| width="250" align="center" | PfR193A (NC)
| align="center" | 13.6
| align="center" | 733.9
| align="center" | 80.9
| align="center" | 9.00
|-
| width="250" align="center" | MvR76 (NC)
| align="center" | 20.2
| align="center" | 1015.0
| align="center" | 64.5
| align="center" | 12.2
|-
| width="250" align="center" | DvR115G (NC)
| align="center" | 10.9
| align="center" | 608.7
| align="center" | 115.6
| align="center" | 6.50
|-
| width="250" align="center" | MrR110B (NC5)
| align="center" | 11.8
| align="center" | 707.0
| align="center" | 99.2
| align="center" | 7.80
|-
| width="250" align="center" | VpR247 (NC5)
| align="center" | 12.5
| align="center" | 661.2
| align="center" | 88.3
| align="center" | 8.05
|-
| width="250" align="center" | BcR147A (NC)
| align="center" | 11.9
| align="center" | 645.0
| align="center" | 104.0
| align="center" | 7.20
|-
| width="250" align="center" | WR73 (NC5)
| align="center" | 21.9
| align="center" | 1261.0 *
| align="center" | 41.3 *
| align="center" | 13.0
|-
| width="250" align="center" | NsR431C (NC5)
| align="center" | 16.8
| align="center" | 855.5
| align="center" | 71.2
| align="center" | 10.6
|-
| width="250" align="center" | StR82 (NC)
| align="center" | 9.2
| align="center" | 537.3
| align="center" | 100.4
| align="center" | 6.6
|}

*data obtained on an 800 MHz Bruker spectrometer at 298 K.

 

== '''Protocols for Bruker and Varian NMR Instruments''' ==

*[[Measuring 15N T1 and T2 Relaxation Times on Bruker Instruments|Bruker protocol]]
*[[Estimation of rotational correlation time|Varian protocol]]

 

== '''References''' ==

<references />

NMR determined Rotational correlation time

2011-10-13T22:36:46Z

Alex:

Creating NOESY peaklists with CARA

2011-04-23T15:57:20Z

Alex: Created page with '# Run 'ExportToCSI' script to generate an input file for CSI # Run CSI program # Run the 'CreateShortSequentialSpinLinks' script to generate spin-links based on CSI prediction # …'

# Run 'ExportToCSI' script to generate an input file for CSI
# Run CSI program
# Run the 'CreateShortSequentialSpinLinks' script to generate spin-links based on CSI prediction
# Remove all unnecessary spins, such as draft assignments (e.g. '?HA'), projected spins (e.g. 'HA-1') and GFT pseudospins (e.g. 'CApCB'). Bulk remove is possible with scripts such as RemoveSins, RemoveProjectedSpins and RemoveGFTSpins.
# Open an HSQC spectrum in PolyScope window with the corresponding NOESY spectrum loaded in the strip. In the 'Strip' menu make sure that the 'Show Spin Links' box is check, and the 'Show Inferred Peaks' is unchecked. Select 'File' -> 'Export...' -> 'Strip Peaks to MonoScope...' to create an initial peaklist with existing assignments.
# Many predicted peaks may not be actually observed. Therefore, integrate the peak list first and then use the PeakListReport script to delete all peaks weaker than a certain threshold.

Automated peak picking

# Define the peak linewidths in the peak model for the peak list of interest.
# Set 'DeafultPeaklist' and 'PeakListToSpectrumMapping' attributes of the corresponding spectrum according to the peaklist.
# Run the PickAll3DNOESY script to automatically pick peaks.
# Run the 'PeakListReport' script to delete diagonal peaks.
# Refine the resulting peaklist by interactivelly scanning all strips.

Resonance Assignment/CARA

2011-04-23T15:38:02Z

Alex:

The topics below provide an overview of resonance assignment of 15N,13C-labeled proteins using CARA. For more information and tutorials please consult [http://www.nmr.ch CARA web site].

*[[CARA Introduction|Introduction]]
*[[CARA vs Xeasy|Differences from XEASY]]
*[[CARA Scopes|Scopes - Displaying NMR Spectra]]

*[[Resonance Assignment/CARA/Starting a new project|Starting a new project]]
*[[Resonance Assignment/CARA/Backbone assignment|Backbone resonance assignment]]
*Side Chain Assignment
**[[Aliphatic Side Chain Assignment with CARA|Aliphatic side-chain assignment]]
**[[Aromatic Side Chain Assignment with CARA|Aromatic side-chain assignment]]
**[[Amide Side Chain Assignment with CARA|Amide side-chain assignment]]
*[[Creating NOESY peaklists with CARA|Creating NOESY peaklists]]

Also see [[Resonance_Assignment/CARA/Backbone_assignment_GFT|backbone assignment with GFT spectra]]