NESG Wiki - User contributions [en]

Processing NMR spectra with PROSA

2021-02-18T19:45:16Z

Krishna:

PROSA is software program for Fourier transformation of NMR data originally developed by Peter Guntert in the lab of Prof. Kurt Wuthrich at ETH Zurich.

For additinal information and licences request see http://www.bpc.uni-frankfurt.de/guentert/wiki/index.php/PROSA

A somewhat outdated manual in PDF format [http://www.bpc.uni-frankfurt.de/guentert/wiki/images/5/56/ProsaManual.pdf]

----
Test 02182021

Main Page

2020-04-29T19:33:05Z

Krishna: /* Equine Gastric Ulcer Syndrome */

__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]]

Main Page

2020-04-29T19:32:40Z

Krishna: /* nmr 2.0 */

__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]]

|}

==Equine Gastric Ulcer Syndrome ==
*[https://equineulcers.online/equine-gastric-ulcer-feeding/ Feeding horses with ulcers]
*[https://equineulcers.online/equine-gastric-ulcer-treatment/ Horse stomach ulcer treatment]

== 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]]

PSVS

2020-04-29T18:33:15Z

Krishna: /* Introduction */

== '''Introduction''' ==

[https://montelionelab.chem.rpi.edu/PSVS/ PSVS] (Protein Structure Validation Server) systematically evaluates the quality of protein structures, and reports a large set of quality scores and constraint analyses [1]. PSVS defines a set of standard quality scores and constraint analyses that are reported for each structure. In addition to experimental constraints, this set encompasses a number of parameters evaluating different aspects of structure quality, including fold and packing, local residue separation, deviations of bond length and bond angles, backbone and side-chain torsion angle conformation, and atomic overlaps. Quality scores are reported based on calibration with a set of high-resolution X-ray crystal structures. A Z-score for the query structure, calculated for the score from each tool, provides an evaluation of the quality of the structure compared to this set of X-ray crystal structures. This server returns a concise validation report that includes a standard set of graphs and tables.  

 

== '''Running the PSVS server''' ==

Once the structure is available, go to the [https://montelionelab.chem.rpi.edu/PSVS/ PSVS server] and upload the input files as described in that page (Figure 1). 

*PDB coordinates: atom names can be in PDB format, DYANA/CYANA or CNS/XPLOR format
*Peak lists (optional, for RPF scores check): separate peak lists from 15N-resolved NOESY and 13C-resolved NOESY.
*Chemical shift (optional, for constraints violation and RPF scores check): BMRB 2.1 or 3.1 format
*Constraints (optional, for constraints violation check): can be in CYANA or XPLOR/CNS format.

 

===== Figure 1:  PSVS 1.4 input page =====

[[Image:PSVS1.4 titlepage.png|800px]]

 

== '''PSVS Output''' ==

One will get an e-mail notice a few minutes after PSVS submisson, and following the link in the e-mail will lead to the PSVS results (Figure 2).  Various outputs can be accessed from tabs in the left margin, and a zipped file of the entire analysis can be downloaded. 

Typical PSVS analyses include:

*Secondary Structure Elements
*Ordered residues
*RMSD values for all residues and ordered residues.
*Ramachandran Plot Summary for ordered residues from Procheck
**Global quality scores for [http://nihserver.mbi.ucla.edu/Verify_3D/ Verify3D], [http://www.came.sbg.ac.at/Services/prosa.html ProsaII], [http://www.biochem.ucl.ac.uk/~roman/procheck/procheck.html Procheck] (phi-psi), Procheck (all) and [http://molprobity.biochem.duke.edu/ MolProbity] Clashscore. It is required that one should never deposit a structure to the PDB with Z scores < -5 for Procheck scores and Molprobity clashscores. General, Z szores > -3 for both Procheck G factor and Molprobity Clashscore are recommended.
**RPF Scores (optional). A good structure should have R > 90%, P > 90%, F> 90% and DP > 70%.

 

===== Figure 2:  PSVS 1.4 output page =====

[[Image:PSVS1.4_outputpage_new2.png|1000px]] 

 

== '''References''' ==

[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795.]

PSVS

2020-04-29T18:27:07Z

Krishna: /* Running the PSVS server */

== '''Introduction''' ==

[http://www-nmr.cabm.rutgers.edu/PSVS/ PSVS] (Protein Structure Validation Server) systematically evaluates the quality of protein structures, and reports a large set of quality scores and constraint analyses [1]. PSVS defines a set of standard quality scores and constraint analyses that are reported for each structure. In addition to experimental constraints, this set encompasses a number of parameters evaluating different aspects of structure quality, including fold and packing, local residue separation, deviations of bond length and bond angles, backbone and side-chain torsion angle conformation, and atomic overlaps. Quality scores are reported based on calibration with a set of high-resolution X-ray crystal structures. A Z-score for the query structure, calculated for the score from each tool, provides an evaluation of the quality of the structure compared to this set of X-ray crystal structures. This server returns a concise validation report that includes a standard set of graphs and tables.  

 

== '''Running the PSVS server''' ==

Once the structure is available, go to the [https://montelionelab.chem.rpi.edu/PSVS/ PSVS server] and upload the input files as described in that page (Figure 1). 

*PDB coordinates: atom names can be in PDB format, DYANA/CYANA or CNS/XPLOR format
*Peak lists (optional, for RPF scores check): separate peak lists from 15N-resolved NOESY and 13C-resolved NOESY.
*Chemical shift (optional, for constraints violation and RPF scores check): BMRB 2.1 or 3.1 format
*Constraints (optional, for constraints violation check): can be in CYANA or XPLOR/CNS format.

 

===== Figure 1:  PSVS 1.4 input page =====

[[Image:PSVS1.4 titlepage.png|800px]]

 

== '''PSVS Output''' ==

One will get an e-mail notice a few minutes after PSVS submisson, and following the link in the e-mail will lead to the PSVS results (Figure 2).  Various outputs can be accessed from tabs in the left margin, and a zipped file of the entire analysis can be downloaded. 

Typical PSVS analyses include:

*Secondary Structure Elements
*Ordered residues
*RMSD values for all residues and ordered residues.
*Ramachandran Plot Summary for ordered residues from Procheck
**Global quality scores for [http://nihserver.mbi.ucla.edu/Verify_3D/ Verify3D], [http://www.came.sbg.ac.at/Services/prosa.html ProsaII], [http://www.biochem.ucl.ac.uk/~roman/procheck/procheck.html Procheck] (phi-psi), Procheck (all) and [http://molprobity.biochem.duke.edu/ MolProbity] Clashscore. It is required that one should never deposit a structure to the PDB with Z scores < -5 for Procheck scores and Molprobity clashscores. General, Z szores > -3 for both Procheck G factor and Molprobity Clashscore are recommended.
**RPF Scores (optional). A good structure should have R > 90%, P > 90%, F> 90% and DP > 70%.

 

===== Figure 2:  PSVS 1.4 output page =====

[[Image:PSVS1.4_outputpage_new2.png|1000px]] 

 

== '''References''' ==

[http://www3.interscience.wiley.com/journal/114029977/abstract 1. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Evaluating protein structures determined by structural genomics consortia. ''Proteins 66'', 778-795.]

TALOS

2014-01-09T19:33:43Z

Krishna: /* Generating TALOS dihedral angle constraints with CYANA (UB) */

== '''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
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 
#: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 constraints 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 />

Buffer optimization

2012-11-26T23:01:28Z

Krishna:

Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues: 

#slow precipitation
#mixture of folded and unfolded protein
#aggregation problems
#multiple populations (too many peaks) for other reasons
#and many other reasons

 

At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by 1H-15N HSQC spectra after exchanging into 13 commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', 46, 11-22.</ref> 

==== '''NMR screening and optimization buffers:''' ====

MJ001        20mM MES, pH 6.5, 100mM NaCl, 5mM CaCl2, 10mM DTT, 50 uM DSS, 0.02% NaN3, 1x Proteinase Inhibitors, 5% D2O

MJ002        20mM NH4OAc, pH 5.5, 100mM NaCl, 5mM CaCl2, 10mM DTT, 50 uM DSS, 0.02% NaN3, 1X Proteinase Inhibitors, 5% D2O 

MJ003        20mM NH4OAc, pH 4.5, 100mM NaCl, 5mM CaCl2, 10mM DTT, 50 uM DSS, 0.02% NaN3, 1X Proteinase Inhibitors, 5% D2O 

MJ004        50mM NH4OAc, pH 5.0, 10mM DTT, 50mM Arginine, 50 uM DSS, 0.02% NaN3, 1X Proteinase inhibitors, 5% D2O

MJ005        50mM NH4OAc, pH 5.0, 10mM DTT, 5% Acetonitrile, 50 uM DSS, 0.02% NaN3, 1X Proteinase inhibitors, 5% D2O

MJ006        50mM MES, pH 6.0, 10mM DTT, 50mM Arginine, 50 uM DSS, 0.02% NaN3, 1X Proteinase inhibitors, 5% D2O

MJ007        50mM MES, pH 6.0, 10mM DTT, 5% Acetonitrile, 50 uM DSS, 0.02% NaN3, 1X Proteinase inhibitors, 5% D2O

MJ008        25mM Na2PO4, pH 6.5, 450mM NaCl, 10mM DTT, 20uM ZnSO4, 50 uM DSS, 0.02% NaN3, 1X Proteinase inhibitors, 5% D2O

MJ009        20mM MES, pH 6.5, 100mM NaCl, 5% Acetonitrile, 10mM DTT, 50 uM DSS, 0.02% NaN3, 1X Proteinase Inhibitors, 5% D2O

MJ010        20mM MES, pH 6.5, 100mM NaCl, 50mM Arginine, 10mM DTT, 50 uM DSS, 0.02% NaN3, 1X Proteinase Inhibitors, 5% D2O

MJ011        20mM MES, pH 6.5, 100mM NaCl, 1% Zwitter, 10mM DTT, 50 uM DSS, 0.02% NaN3, 1X Proteinase Inhibitors, 5% D2O

MJ012        20mM MES, pH 6.5, 100mM NaCl,  50uM ZnSO4, 10mM DTT, 50 uM DSS, 0.02% NaN3, 1X Proteinase Inhibitors, 5% D2O

Low Salt     10mM Tris-HCl, pH 7.5, 5mM DTT, 100mM NaCl , 0.02% NaN3, 50 uM DSS, 1X Proteinase Inhibitors, 5% D2O 

 

==== '''Abbreviations:''' ====

DTT: Dithiothreitol

Zwitter: ZWITTERAGENT® 3-12 Detergent [cat.693015](CALBIOCHEM)

MES: 2-(''N''-morpholino)ethanesulfonic acid

Tris: tris(hydroxymethyl)aminomethane;

Protease inhibitors: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE);

DSS: 2,2-Dimethyl-2-silapentane-5-sulfonic acid

==== References ====

<references />

File:IMG 1464.JPG

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File:Keith Hamilton.ppt

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File:Yuefeng Tang.ppt

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File:Xianzhong Xu.ppt

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File:Roberto Tejero.ppt

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File:RamaKrishna 2.ppt

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File:Mike Kennedy.pdf

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File:Jeff Mills.ppt

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File:RamaKrishna 1.ppt

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File:Asli Ertekin.ppt

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File:Guy Montelione 2.ppt

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File:Guy Montelione 1.ppt

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File:Gaohua Liu.ppt

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File:Bin Wu.ppt

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File:Binchen Mao.ppt

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File:Ali Yilmaz.ppt

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File:Alex Lemak.ppt

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