NESG Wiki - User contributions [en]

Homodimer Structure Calculation with Symmetry Constraints

2009-12-15T21:22:11Z

Prossi:

Structure Calculation With RDC's Using CYANA

2009-12-15T21:21:16Z

Prossi:

== '''Introduction''' ==

'''IMPORTANT DISCLAIMER:''' A number of NESG NMR groups are currently beta-testing sites for CYANA-3.0 and the information in this page is intended for use by the licensed members of the NESG consortium, other beta testers, and is to be used in accordance to the program licensing agreement.

 

The following page describes the setup of and analysis of an automated structure determination starting from NOEs peaklists and residual dipolar coupling (RDC) constraints in the framework of CYANA 3.0. 

CYANA version 3.0 incorporates many new features including: 

a) inclusion of residual dipolar coupling in structure calculation (RDC) 

b) inclusion of pseudocontact shifts from paramagnetic centers in structure calculation (PCS) 

c) improved function for symmetric dimers and annotation of intermolecular NOE contacts in the peaklist using the xeasy color code notation.   

Good agreement between global orientational constraints from RDC and distance information from NOEs is very important to achieve better structures by NMR.  Results should always be accompanied by energy refinement in CNS or NIH-XPLOR.  Further information about the program and publication references can be found in the [http://www.cyana.org/wiki/index.php/Main_Page CYANA WIKI] page. 

 

== Automated NOE and RDC and Structure Calculation Setup ==

The structure calculation with automated noesy assignments and RDC restraints follows the canonical CYANA recipe.  Simple annealing runs starting from a set of constraints that include RDC are easily derived by simplifying the scripts below and following the demo scripts. The program requires a sequence file (name.seq), a proton assignment list (name.prot), a noesy peaklist set (name.peaks), an RDC list (name.rdc), a CALC.cya script and an init.cya script.   

==== Sequence file ====

The sequence file now includes the RDC tensor origin separated by dummy linker residues (LL5): 
<pre>MET 1
THR 2
SER 3
THR 4
PHE 5
ASP 6
ARG 7
VAL 8
ALA 9
THR 10

PL 350
LL5 351
LL5 352
LL5 353
LL5 354
LL5 355
ORI 360

</pre>
 

==== RDC constraint file ====

The RDC list supports multiple interatomic vectors in multiple media. RDC with distinct scaling factors and distinct ORI residue numbers are listed in a single file. The program supports the Da (magnitude) and R (Rhombicity) notation typical of programs such as PALES, REDCAT etc. Below is a sample RDC file that includes N-H, N-CA (intra), and N-C' (sequential) vectors in one medium with adequate error (here the following errors were used:  ~10 % the RDC spread for N-H vectors, the error determined by analysis of ''J''-modulated experiments for N-CA and N-C' RDC measurement) and [http://www.ncbi.nlm.nih.gov/pubmed/18388951 weight factors]: 
<pre># Orientation Magnitude Rhombicity ORI residue number
1 5.39535 0.63125 360
# First atom Second atom RDC Error Weight Orientation
7 ARG H 7 ARG N 5.936 2.000 1.000 1
8 VAL H 8 VAL N 3.827 2.000 1.000 1
9 ALA H 9 ALA N -2.822 2.000 1.000 1
10 THR H 10 THR N -0.674 2.000 1.000 1
11 ILE H 11 ILE N 4.945 2.000 1.000 1
12 ILE H 12 ILE N 1.709 2.000 1.000 1
13 ALA H 13 ALA N -1.336 2.000 1.000 1
#
4 THR N 3 SER C 1.267 0.095 8.330 1
5 PHE N 4 THR C -0.246 0.207 8.330 1
7 ARG N 6 ASP C 0.161 0.052 8.330 1
8 VAL N 7 ARG C 0.439 0.034 8.330 1
9 ALA N 8 VAL C -0.076 0.040 8.330 1
10 THR N 9 ALA C -0.957 0.048 8.330 1
11 ILE N 10 THR C 1.123 0.022 8.330 1
12 ILE N 11 ILE C -0.440 0.037 8.330 1
13 ALA N 12 ILE C 0.065 0.026 8.330 1
#
3 SER N 3 SER CA 0.251 0.199 8.330 1
4 THR N 4 THR CA -0.265 0.258 8.330 1
5 PHE N 5 PHE CA -0.499 0.281 8.330 1
7 ARG N 7 ARG CA -0.457 0.200 8.330 1
8 VAL N 8 VAL CA -0.481 0.154 8.330 1
9 ALA N 9 ALA CA 0.349 0.083 8.330 1
10 THR N 10 THR CA 0.548 0.121 8.330 1
11 ILE N 11 ILE CA -0.091 0.111 8.330 1
12 ILE N 12 ILE CA -0.678 0.078 8.330 1
13 ALA N 13 ALA CA 1.014 0.108 8.330 1</pre>
Multiple media (e.g. orientations) should be listed as follows: 
<pre># Orientation Magnitude Rhombicity ORI residue number
1 5.39535 0.63125 360
2 7.55656 0.58200 370
</pre>
and the sequence should be modified to include further links and ORI: 
<pre>ALA 9
THR 10

PL 350
LL5 351
LL5 352
LL5 353
LL5 354
LL5 355
ORI 360
LL5 361
LL5 362
LL5 363
LL5 364
LL5 365
ORI 370</pre>

==== Initial model-free determination of Da and R from assigned RDCs ====

If the values of Da and R are not known they can be determined using any desired software OR using the FindTensor.cya script below. The program yields results that are equivalent to PALES assiuming the same fitting method is employed. 
<pre>## 8DEMOS: FindTensor - Determine alignment tensor
##
## Determine magnitude and rhombicity of the alignment tensor
## from input RDCs

# determine tensor from histogram, no structure needed

read rdc phage_all_mono.rdc
print "    Input alignment tensor:"
do i 1 orientations
  print "    Orientation $i: magnitude = $magnitude(i) Hz, rhombicity = $rhombicity(i)."
end do

rdc fittensor method=simplex       # (can take several minutes)
#rdc fittensor method=gridsearch   # systematic search (very slow)

# alternatively, determine tensor from given structure by SVD

read rdc phage_all_mono.rdc
read pdb final.pdb
overview

</pre>
The dummy values in the rdc list are read initially and they can be updated after running the FindTensor.cya routine. If no models are present (e.g. final.pdb) the program will terminate with a warning. 

==== The CALC.cya file ====

The working directory that contains all the files necessary to start the calcualtion is ready and the CALC.cya modified for the presence of RDC constraints is used: 
<pre>peaks  := ali5.peaks,aro5.peaks,n3.peaks # names of NOESY peak lists
prot  := RpR324.prot # names of chemical shift lists
restraints  := ssa.cya,phage_all_mono.rdc # additional (non-NOE) constraints
tolerance  := 0.04,0.025,0.3 # chemical shift tolerances: HX2-HX1-X1
calibration := # NOE calibration parameters
structures  := 100,20 # number of initial, final structures
steps  := 10000 # number of torsion angle dynamics steps
rmsdrange  := 10..80 # residue range for RMSD calculation
randomseed  := 56231 # random number generator seed

weight_rdc = 0.02 # weight for RDC restraints
cut_rdc = 0.2 # cutoff for RDC violation output

ssa
noeassign peaks=$peaks prot=$prot autoaco

</pre>
Notice that the 'restraints' row contain the forced stereospecifically assigned methyls and sidechain NH's and the .rdc file.  Also, the NOE vs. RDC weight is set by the weight_rdc and cut_rdc functions.  The remaining instructions are identical to the CYANA-2.1 file. 

==== The init.cya file ====

Further parameters are specified in the init.cya file below: 
<pre>name:=RpR324
rmsdrange:=10-80
cyanalib
nproc:=8
read seq $name.seq
rdcdistances</pre>
The above script is intended to run off a single dual-quad core machine (nproc=8). 

===== rdcdistances.cya file =====

Please note the rdcdistance.cya macro is being called by the init.cya setup file.  This file, located in the cyana-3.0/macro directory contains the supported RDC vectors, more vectors could potentially be added such as Trp Nε1-Hε1 that maybe useful in deuterated sample to direct the large hydrophobic sidechain.  
<pre># Copyright (c) 2002-08 Peter Guntert. All rights reserved.
## 7MACROS: rdcdistances - CYANA macro
##
## Parameters: (none)
##
# dipole definition format: atom1_name atom2_name atom1_index atom2_index
# if indexes are missing, zeros are assumed

var info echo

syntax

info:=none; echo:=off
rdc distance "N  H"    distance=1.041
rdc distance "CA HA"   distance=1.117
rdc distance "C  CA"   distance=1.525
rdc distance "C  N"    distance=2.461
rdc distance "C  N -1" distance=1.329
rdc distance "CA N"    distance=1.458
rdc distance "CA N -1" distance=2.425
rdc distance "CA H"    distance=2.117
rdc distance "CA H -1" distance=2.533
rdc distance "C  H -1" distance=2.000
rdc distance "C  HA"   distance=2.144
rdc distance "CB HB"   distance=1.080
rdc distance "CA CB"   distance=1.532
unset info
print "    Standard RDC distances defined."
</pre>

==== Notes on run execution ====

The command line execution form single machine or cluster using MPI implememntation (highly recommended) is carried on as usual: 
<pre>/cyana-3.0/cyana CALC > & log &
</pre>
the MPI is launched using the script called, for example, submit_cyana: 
<pre>#!/bin/bash
#PBS -S /bin/bash
#PBS -N cyana
#PBS -lnodes=6:ppn=8
lamboot ~/bhost.def
cd /farm/users/prossi/RpR324_structure/cyana_new_mono2
/opt/openmpi/tcp-gnu/bin/mpirun /farm/software/cyana-3.0-mpi/cyana CALC.cya
lamhalt</pre>
 with the command: 
<pre>qsub -q @master3 submit_cyana
</pre>
The starting scripts are highly system specific they are almost guaranteed NOT to work on your sytem and are given here for general information only. 

 

== Output analysis ==

The output analysis is carried out in the usual manner, it should be noted that, during the calculation the specified values Da and R are kept fixed.  Following the final cycle a new model-based estimate of Da and R is calculated and used to compute the RDC violations and their contribution to the target function and the quality factor (Q). The resulting target function will be increased by the number and extent of RDC violations in addition to other violations from dihedral, vdw, and NOEs restraints.

A partial output file is given below (final.ovw): 
<pre>
Structural statistics:

str target upper limits van der Waals RDCs
function # rms max # sum max # rms max
1 8.98 5 0.0089 0.40 12 18.5 0.37 13 0.2508 2.27
2 8.96 7 0.0083 0.31 15 17.4 0.38 19 0.2656 2.27
3 9.69 11 0.0122 0.54 15 18.6 0.38 18 0.2595 2.28
4 9.63 6 0.0068 0.20 16 18.9 0.38 18 0.2604 2.27
5 9.41 14 0.0089 0.22 17 20.4 0.37 14 0.2521 2.26
6 9.80 10 0.0085 0.24 17 19.7 0.38 15 0.2563 2.27
7 10.52 12 0.0158 0.78 15 19.1 0.38 13 0.2554 2.29
8 10.05 10 0.0084 0.18 18 20.3 0.42 18 0.2587 2.28
9 10.57 7 0.0079 0.29 19 18.3 0.53 16 0.3169 2.43
10 10.38 13 0.0088 0.21 22 20.5 0.37 21 0.2636 2.28
11 10.33 7 0.0067 0.16 17 19.6 0.64 15 0.2663 2.28
12 9.93 9 0.0090 0.29 19 20.6 0.38 20 0.2541 2.27
13 10.12 12 0.0098 0.23 21 19.9 0.47 13 0.2681 2.30
14 10.53 7 0.0077 0.19 23 19.3 0.38 11 0.3145 2.40
15 10.96 8 0.0089 0.31 21 22.6 0.38 12 0.2639 2.28
16 10.50 12 0.0100 0.29 23 20.1 0.37 15 0.2696 2.27
17 10.56 14 0.0119 0.30 23 21.1 0.37 16 0.2736 2.33
18 10.75 17 0.0146 0.60 21 20.0 0.37 12 0.2547 2.27
19 10.88 18 0.0125 0.38 19 23.3 0.38 10 0.2506 2.30
20 10.88 8 0.0083 0.26 23 21.4 0.42 15 0.2644 2.28

Ave 10.17 10 0.0097 0.32 19 20.0 0.41 15 0.2660 2.29
+/- 0.59 4 0.0024 0.15 3 1.4 0.07 3 0.0177 0.04
Min 8.96 5 0.0067 0.16 12 17.4 0.37 10 0.2506 2.26
Max 10.96 18 0.0158 0.78 23 23.3 0.64 21 0.3169 2.43
Cut 0.10 0.20 0.20

Constraints violated in 6 or more structures:
# mean max. 1 5 10 15 20
Upper HA PRO 19 - HB3 ARG 20 5.50 15 0.11 0.21 +++++++ ++++ ++*+ peak 970
Upper HA ILE 23 - QB SER 27 5.34 6 0.09 0.16 + ++ + +* peak 276
Upper HA ILE 30 - HB2 LEU 33 4.95 10 0.10 0.15 ++++ ++ ++ +* peak 313
VdW CB ALA 69 - H THR 70 2.55 18 0.24 0.28 ++++++++ ++++ *+++++
VdW O THR 71 - N PHE 75 2.75 12 0.20 0.32 ++* +++ + ++ +++
VdW O PHE 75 - C VAL 76 2.80 13 0.21 0.31 ++++ + *+++++++
VdW CG1 VAL 76 - HG2 LYS 78 2.60 9 0.17 0.25 ++* +++++ +
VdW HG3 LYS 78 - C LYS 78 2.50 14 0.22 0.36 +++*++ +++++ + + +
Ori 1 N ALA 69 - CA ALA 69 -2.84 20 1.95 2.04 +++++++++++++*++++++
Ori 1 H ALA 69 - N ALA 69 -3.32 20 2.01 2.33 ++++++++++++++++*+++
Ori 1 N THR 70 - C ALA 69 0.25 16 0.31 1.06 ++++++++*+++ + + ++
Ori 1 H ASN 79 - N ASN 79 -4.71 10 0.17 0.32 + *+ + ++++ + +
Ori 1 H GLY 92 - N GLY 92 -5.54 8 0.15 0.51 ++++* + ++
Ori 1 H LEU 94 - N LEU 94 0.42 6 0.14 0.54 +*+ +++
3 violated distance restraints.
5 violated van der Waals restraints.
6 violated residual dipolar coupling restraints.

RDC statistics:
Correlation coefficient  : 0.906 +/- 0.003 (0.899..0.909, best in conformer 4)
Q = rms(Dcalc-Dobs)/rms(Dobs): 42.709 +/- 0.583 % (42.073..44.194)
Q normalized by tensor  : 32.943 +/- 0.638 % (32.231..34.776)
Alignment tensor magnitude  : 5.881 +/- 0.045 Hz (5.760..5.950, best 5.898; input 5.898)
Alignment tensor rhombicity  : 0.537 +/- 0.007 (0.524..0.556, best 0.539; input 0.539)

</pre>
 

 

 

-- PaoloRossi - 14 Dec 2009

2009-11-23T21:22:32Z

Prossi:

AVS

2009-11-23T21:21:29Z

2009-11-23T20:55:02Z

Wiki Tree Layout

2009-11-20T19:38:32Z

Prossi:

This outline of the NESG NMR Wiki is designed to expand on the existing "Master Recipe" and should serve as an experience harvesting tool.

*It has a rather broad coverage to facilitate long-tewrm growth and development. Aditional compact aggregator pages may be needed to pesent specific information concisely.
*There would be separate webs within the wiki: Public(or Main), NESG, and member lab webs. Most common knowlege topics should be public, unless they are specific to NESG
*We assume that the target audience has some knowledge about NMR and protein structure determination, but make the content useful for training
*"Resonance Assignment" and "Structure Determination" chaptes would focus on individual software packages. The XEASY resonance assignment tree, as the most complete, would serve as a template for other software.
*Most chapters should include a "general principles" section.

Please leave your comments/suggestion at the bottom of this page

 

= HTP NMR structure determination =

== Protein Target Selection, Sample Preparation, and Initial Screening ==

#[[Target selection|NESG target selection]] - overview of target selection in PSI III
#[[Bioinformatics with protein sequence]]
#[[DNA cloning protocols|DNA cloning protocols]] 
#[[Protein purification|Protein expression and purification protocols]]  
#Sample Optimization
##[[construct optimization]]
##[[Buffer optimization]]
##[[Cofactor optimization]]
#Initial protein analysis
##[[SDS page gel]]
##[[Protein concentration|Protein concentration measurements]]
##[[Oligomerization Status|Assessment of Oligomerization Status]]
###[[Gel filtration and light scattering|gel-filtration and light scattering]]
###[[Sedimentation equilibrium|Sedimentation equilibrium]]
###[[NMR determined Rotational correlation time]]
##[[MassSpectrometry|Mass spectrum]]
##NMR screening
###[[1D screening|1H 1D screening]]
###[[Nhsqc screen|Initial [15N,1H] HSQC]] 

== NMR Data Collection ==

#Routine operation
##[[NMR sample tubes]]
##[[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]]
##Custom NMR experiment setup scripts for VNMRJ
##1D 1H NMR spectra and 2D [15N, 1H]-HSQC
##[[Estimation of rotational correlation time]]
##Estimation of measurement time
##NMR experiments for spin system identification
##2D and 3D NOESY
##Double and triple NMR experiments
###3D CBCA(CO)NH and HNCACB
###3D HNCA and HN(CO)CA
###3D HAHB(CO)NH
###(4,3)D CABCA(CO)NH and HNCACB
###(4,3)D HABCAB(CO)NH
###(H)CCH
###(H)CCH-TOCSY
###H(C)CH
###H(C)CH-TOCSY
###(4,3)D HCCH
##Other NMR experiments
###2D [13C, 1H]-HSQC for 5% 13C-labeled samples
###2D [15N, 1H]-long-range-HSQC for determination of histidine protomer state
###MEXICO
###CLEANEX
###H-D exchange experiment
###15N spin relaxation parameters
#Advanced problems for data collection
##[[Setting up non-uniformly sampled spectra]]
###[[NUS giude for Varian|Guide for Varian/BioPack]]
###[[NUS guide for Bruker according to Arrowsmith group in Toronto|Guide for Bruker/Topspin according to Arrowsmith group]]
#Maintenance
##VARIAN
###Installing and updating BioPack
###Full Probefile calibration
###Rebooting the console
###Cryoprobe conditioning
##BRUKER

== NMR Data Processing ==

#General Priciples and Concepts
##Fourier transformation
###Zero-filling
###Apodization
###Phasing
###Linear prediction
###G-matrix Fourier transformation (GFT)
##Alternatives to Fourier transformation
###Maximum entropy reconstruction
###MDD reconstruction
###...
#Practical Aspects
##NMRPIPE
###General information
###Buffalo's Processing Protocol using NMRpipe
##PROSA
##TOPSPIN
##[[AGNuS/AutoProc|AGNuS/AutoProc]]
##UBNMR
##Spectral format conversion

 

== Resonance Assignment ==

This chapter would focus on individual data analysis and resonance assignment packages, as most people stick to a particular software for entire structure determination projects.

 

#[[Principles and concepts]]
##[[Stable isotope labeling schemes]]
##[[NMR experiments]]
###[[Through-bond]]
###[[Through space]]
##[[Spin systems]]
###[[Definitions]]
###[[Identification]]
###[[Linking spin systems]]
###[[Matching onto covalent structure]]
#[[Practical aspects]]
##[[Semi-automated protocols]]
###[[CARA]]
####[[Spin System Identification with CARA|Spin System Identification in 2D 15N-HSQC and 3D HNNCO]]
####[[Backbone Assignment with CARA|Backbone Resonance Assignment]]
####[[HA and HB Assignment with CARA|Assignment of HA and HB Resonances with (4,3)D GFT HABCAB(CO)NHN]]
####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]]
###[[Sparky]]
###[[XEASY]]
####[[XEASY Spin system identification|Spin system identification]]
####[[XEASY Backbone Assignment|Backbone resonance assignment]]''' '''
#####GFT-based spectra
######[[HNCACAB/CABCA(CO)NH]]
#####Conventional spectra
######[[HNCACB/CBCA(CO)NH]]
######HNCA/HN(CO)CA
######HNCO/HN(CA)CO
######NOESY/TOCSY
####[[XEASY Side Chain Assignment|Side chain resonance assignment]]
#####Aliphatic
######GFT NMR spectra
#######[[HA and HB Assignment with GFT in XEASY|(4,3)D GFT HABCAB(CO)NHN]]
#######[[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|(4,3)D GFT HCCH]]
######Conventional spectra
#######HAHB(CO)NH
#######HCCH-COSY
#######HCCH-TOCSY
#######[[Side chain assignment with CN-NOESY in XEASY|Simultaneous NOESY]]
#######(H)CC-TOCSY-(CO)NH
#######H(CC-TOCSY-CO)NH
#####[[Aromatic side chain assignment with Aro-HCCH-COSY in XEASY|Aromatic]]
######GFT-based spectra
######Conventional spectra
#####Other
######Trp e1 NH and d1 CH
######[[Met methyl assignment with NOESY|Met e CH3 ]]
######[[Amide Side Chain assignment with NOESY|Asn d2 and Gln e2 NH2]]
#####NOESY peak integration
##Automated protocols
###[[AutoAssign|AutoAssign]]
###[[AutoAssign WebServer|AutoAssign server]]
###[[Abacus|ABACUS]]
###[[The PINE Server|PINE server]]
##Validation of resonance assignment
###[[AVS|Assignment validation suite (AVS)]]
###[[LACS|Linear analysis of chemical shift (LACS)]]
##Depositing chemical shifts

 

== Structure Calculation and Validation ==

#[[Structure Calculation and Validation|Principles and concepts]]
#Practical aspects
##Structure calculation
###CYANA
####[[CYANA|Getting started]]
####[[FOUND|FOUND]]
####[[TALOS|TALOS]]
####[[GLOMSA|GLOMSA]]
####[[NOE Calibration Using CYANA|NOE calibration]]
####[[Manual Structure Calculation Using CYANA|Manual structure calculation]]
####[[Automated NOESY Assignment Using CYANA|Automated NOESY assignment and structure calculation]]
####[[Structure Calculation With RDC's Using CYANA|Structure calculation with residual dipolar couplings]] (link to REDCAT/PALES,FINDTENSOR, .rdc file, adding ORI to PDB file)
####[[Homodimer Structure Calculation Using CYANA|Homodimer structure calculations]][[Homodimer Structure Calculation Using CYANA| ]]
###AutoStructure
####[[AutoStructure/RPF Theory|Theory]]
####[[AutoStructure|Getting started]]
####[[CYANA Structure Calculations Using AutoStructure|CYANA run]]
####[[XPLOR Structure Calculations Using AutoStructure|XPLOR run]]
####[[Analyzing AutoStructure Output Directories|Analyzing the output]]
####[[RPF Analysis|RPF/DP scores]]
####[[Structure Calculation Using AS-DP|Structure calculation using AS-DP]]
###"Consensus" Approaches
####[[Overview of Consensus Runs|Overview of Consensus runs]]
####[[Finding Consensus NOE Assignments|Finding Consensus NOE assignments]]
####[[Validation of Consensus Run|Validation of Consensus runs]]
###[[Structure Calculation Using CS-Rosetta|CS-ROSETTA]]
###[[Structure Calculation Using CS-CP ROSETTA|CS-DP ROSETTA]]
###[[Structure Calculation Using RDC-ROSETTA|RDC-ROSETTA]]
###[[RDC-Assisted Dimer Structure Determination|RDC-assisted dimer structure calculation]] 
###Special topics
####[[Protein-Ligand Complexes|Protein-Ligand complexes]]
####[[Working With Metal Ions|Metal ions]]
####[[Working With Dimers|Dimers]]
####[[Residual Dipolar Couplings in Structure Refinement|Residual Dipolar Couplings]]
####[[REDCAT|REDCAT]] and [[REDCRAFT|REDCRAFT]]
####[[Paramagnetic Constraints in Structure Determination|Paramagnetic constraints]]
##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]]

-- JeffMills - 28 May 2009

Here are two comments from Guy:

- need to have centralized site for downloading all software that NESG has developed or licensed; this would be a central site for NESG scientists to use to access the latest version of all software

- need to allow outside users to access links to all software (they will need licenses to download) and also to download software from NESG

-- AlexEletski - 13 Jul 2009

LACS

2009-11-20T19:22:24Z

Prossi:

== '''Introduction''' ==

Linear analysis of chemical shift ([http://www.ncbi.nlm.nih.gov/pubmed/16041479 LACS]) provides an independent means for absolute [[media:iupac.pdf|referencing]] of protein chemical shifts.  LACS is routinely run on all chemical shift sets submitted to the BioMagResBank (BMRB). 

In the contest of protein structure determination chemical shifts (CS) play a key role as reporters of secondary structure.  Backbone chemical shift derived dihedral angles can be obtained through programs such as [http://www.ncbi.nlm.nih.gov/pubmed/19548092 TALOS+] and are used as constraints in the structure determination process. The backbone shift set (CA, CB, CO, HN, N, HA) is also used in the molecular fragment selection (MFR) for [http://www.ncbi.nlm.nih.gov/pubmed/18326625 CS-Rosetta] structure prediction protocol.  Here fragments are selected from the protein databank (PDB) that have the same predicted CS as the query sequence.  Successful use of chemical shift for secondary structure inference requires the correct referencing.  The LACS server gives independent CS offsets for CA, CB, CO and HA and allows for effective correction of chemical shift sets derived from [''U''-13C,15N] and from [''U''-2H,13C,15N].  The latter labeling scheme may show distinct degrees of offset for CA and CB due to the different number of bound deuterons which affect the value of the carbon to different degree.   

 

== '''Running LACS''' ==

LACS can be run on the native [http://bija.nmrfam.wisc.edu/MANI-LACS/ server] using an edited version of the chemical shift file in bmrb 2.1 format, the user's email and project name.  The server returns the results very rapidly, the chemical shift file format is stringent and the output is of straight forward interpretation. 

The items returned are:

i) a text file with the nucleus specific offsets from the linear fit of the secondary CS  (e. g. deltaCA vs. deltaCA-deltaCB), obtained by subtracting the random coil value from the atom of interest.

ii) plots of the secondary CS outliers that may reveal incorrectly assigned residues.

 

A running [[Media:WR73-ILVFY_111809_4LACS.bmrb|bmrb]] file and text [[Media:LACS_output.txt|output]] example are provided for user's convenience, adhering to the format should provide smooth operation of the server. 

 

=== Example BMRB input for LACS ===
<pre> _Mol_residue_sequence
;
MLIYKDIFTDDELSSDSFPM
KLVDDLVYEFKGKHVVRKEG
EIVLAGSNPSAEEGAEDDGS
DEHVERGIDIVLNHKLVEMN
CYEDASMFKAYIKKFMKNVI
DHMEKNNRDKADVDAFKKKI
QGWVVSLLAKDRFKNLAFFI
GERAAEGAENGQVAIIEYRD
VDGTEVPTLMLVKEAIIEEK
CLE
;

loop_
_Residue_seq_code
_Residue_label
_Chem_shift_ambiguity_code
4 1 MET C C 170.742 . 1
5 1 MET CA C 54.828 . 1
6 1 MET CB C 32.579 . 1
7 2 LEU H H 8.934 . 1
8 2 LEU HD1 H 0.688 . 1

</pre>
 

=== Example LACS ouput ===

The CA correction of -0.19 ppm is found (last line).
<pre>data_LACS

#################################
# LACS Output Information #
#################################

############################################################
# LACS Designator Definition #
# #
# Index Value Definition #
# #
# 0 Outliers #
# 1 Normal points #
# #
############################################################

save_LACS_CACB_CA_output
_LACS_plot.Sf_category LACS_output
_LACS_plot.Input_file_name "data.txt"

_LACS_plot.X_coord_name CA-CB
_LACS_plot.Y_coord_name CA
_LACS_plot.Line_1_terminator_val_x_1 -6.35
_LACS_plot.Line_1_terminator_val_y_1 -2.37
_LACS_plot.Line_1_terminator_val_x_2 1.99
_LACS_plot.Line_1_terminator_val_y_2 0.93
_LACS_plot.Line_2_terminator_val_x_1 -2.00
_LACS_plot.Line_2_terminator_val_y_1 -1.18
_LACS_plot.Line_2_terminator_val_x_2 5.76
_LACS_plot.Line_2_terminator_val_y_2 4.33
_LACS_plot.Y_axis_chem_shift_offset -0.19

</pre>
 

== '''Practical Experience with Running LACS in the NESG''' ==

In the NESG the IUPAC referencing is obtained using internal 50 uM DSS and setting the DSS methyl peak to 0.00 ppm at the temperature of interest.  The spectrometer temperature is calibrated using a neat methanol sample in the 10 - 40 °C range.  Under these conditions, backbone CS of double labeled protein samples derived from triple resonance experiments alone typically show small deviations 0.1 - 0.3 ppm in 13C and 0.01 - 0.03 ppm in 1H.  The final CS set derived from double labeled samples filtered through higher resolution [1H,13C]-HSQC tends to give offsets at the lower extreme of this range.  On a smaller statistical sampling of triply labeled proteins we found higher offset values (0.4 - 0.8 ppm) in CA and CB.  Offsett correction prior to TALOS or MFR runs are therefore highly recommended especially for the treatment of triply labeled samples.  

  

 

editing of this entry is in progress (prossi) 

 

 

-- PaoloRossi - 20 Nov 2009

File:Iupac.pdf

2009-11-20T19:21:57Z

Prossi:

LACS

2009-11-20T19:20:11Z

Prossi:

== '''Introduction''' ==

Linear analysis of chemical shift ([http://www.ncbi.nlm.nih.gov/pubmed/16041479 LACS]) provides an independent means for absolute [[www.bmrb.wisc.edu/iupac.pdf|referencing]] of protein chemical shifts.  LACS is routinely run on all chemical shift sets submitted to the BioMagResBank (BMRB). 

In the contest of protein structure determination chemical shifts (CS) play a key role as reporters of secondary structure.  Backbone chemical shift derived dihedral angles can be obtained through programs such as [http://www.ncbi.nlm.nih.gov/pubmed/19548092 TALOS+] and are used as constraints in the structure determination process. The backbone shift set (CA, CB, CO, HN, N, HA) is also used in the molecular fragment selection (MFR) for [http://www.ncbi.nlm.nih.gov/pubmed/18326625 CS-Rosetta] structure prediction protocol.  Here fragments are selected from the protein databank (PDB) that have the same predicted CS as the query sequence.  Successful use of chemical shift for secondary structure inference requires the correct referencing.  The LACS server gives independent CS offsets for CA, CB, CO and HA and allows for effective correction of chemical shift sets derived from [''U''-13C,15N] and from [''U''-2H,13C,15N].  The latter labeling scheme may show distinct degrees of offset for CA and CB due to the different number of bound deuterons which affect the value of the carbon to different degree.   

 

== '''Running LACS''' ==

LACS can be run on the native [http://bija.nmrfam.wisc.edu/MANI-LACS/ server] using an edited version of the chemical shift file in bmrb 2.1 format, the user's email and project name.  The server returns the results very rapidly, the chemical shift file format is stringent and the output is of straight forward interpretation. 

The items returned are:

i) a text file with the nucleus specific offsets from the linear fit of the secondary CS  (e. g. deltaCA vs. deltaCA-deltaCB), obtained by subtracting the random coil value from the atom of interest.

ii) plots of the secondary CS outliers that may reveal incorrectly assigned residues.

 

A running [[Media:WR73-ILVFY_111809_4LACS.bmrb|bmrb]] file and text [[Media:LACS_output.txt|output]] example are provided for user's convenience, adhering to the format should provide smooth operation of the server. 

 

=== Example BMRB input for LACS ===
<pre> _Mol_residue_sequence
;
MLIYKDIFTDDELSSDSFPM
KLVDDLVYEFKGKHVVRKEG
EIVLAGSNPSAEEGAEDDGS
DEHVERGIDIVLNHKLVEMN
CYEDASMFKAYIKKFMKNVI
DHMEKNNRDKADVDAFKKKI
QGWVVSLLAKDRFKNLAFFI
GERAAEGAENGQVAIIEYRD
VDGTEVPTLMLVKEAIIEEK
CLE
;

loop_
_Residue_seq_code
_Residue_label
_Chem_shift_ambiguity_code
4 1 MET C C 170.742 . 1
5 1 MET CA C 54.828 . 1
6 1 MET CB C 32.579 . 1
7 2 LEU H H 8.934 . 1
8 2 LEU HD1 H 0.688 . 1

</pre>
 

=== Example LACS ouput ===

The CA correction of -0.19 ppm is found (last line).
<pre>data_LACS

#################################
# LACS Output Information #
#################################

############################################################
# LACS Designator Definition #
# #
# Index Value Definition #
# #
# 0 Outliers #
# 1 Normal points #
# #
############################################################

save_LACS_CACB_CA_output
_LACS_plot.Sf_category LACS_output
_LACS_plot.Input_file_name "data.txt"

_LACS_plot.X_coord_name CA-CB
_LACS_plot.Y_coord_name CA
_LACS_plot.Line_1_terminator_val_x_1 -6.35
_LACS_plot.Line_1_terminator_val_y_1 -2.37
_LACS_plot.Line_1_terminator_val_x_2 1.99
_LACS_plot.Line_1_terminator_val_y_2 0.93
_LACS_plot.Line_2_terminator_val_x_1 -2.00
_LACS_plot.Line_2_terminator_val_y_1 -1.18
_LACS_plot.Line_2_terminator_val_x_2 5.76
_LACS_plot.Line_2_terminator_val_y_2 4.33
_LACS_plot.Y_axis_chem_shift_offset -0.19

</pre>
 

== '''Practical Experience with Running LACS in the NESG''' ==

In the NESG the IUPAC referencing is obtained using internal 50 uM DSS and setting the DSS methyl peak to 0.00 ppm at the temperature of interest.  The spectrometer temperature is calibrated using a neat methanol sample in the 10 - 40 °C range.  Under these conditions, backbone CS of double labeled protein samples derived from triple resonance experiments alone typically show small deviations 0.1 - 0.3 ppm in 13C and 0.01 - 0.03 ppm in 1H.  The final CS set derived from double labeled samples filtered through higher resolution [1H,13C]-HSQC tends to give offsets at the lower extreme of this range.  On a smaller statistical sampling of triply labeled proteins we found higher offset values (0.4 - 0.8 ppm) in CA and CB.  Offsett correction prior to TALOS or MFR runs are therefore highly recommended especially for the treatment of triply labeled samples.  

  

 

editing of this entry is in progress (prossi) 

 

 

-- PaoloRossi - 20 Nov 2009

LACS

2009-11-20T19:18:58Z

Prossi:

== '''Introduction''' ==

Linear analysis of chemical shift ([http://www.ncbi.nlm.nih.gov/pubmed/16041479 LACS]) provides an independent means for absolute [[Www.bmrb.wisc.edu/iupac.pdf|referencing]] of protein chemical shifts.  LACS is routinely run on all chemical shift sets submitted to the BioMagResBank (BMRB). 

In the contest of protein structure determination chemical shifts (CS) play a key role as reporters of secondary structure.  Backbone chemical shift derived dihedral angles can be obtained through programs such as [http://www.ncbi.nlm.nih.gov/pubmed/19548092 TALOS+] and are used as constraints in the structure determination process. The backbone shift set (CA, CB, CO, HN, N, HA) is also used in the molecular fragment selection (MFR) for [http://www.ncbi.nlm.nih.gov/pubmed/18326625 CS-Rosetta] structure prediction protocol.  Here fragments are selected from the protein databank (PDB) that have the same predicted CS as the query sequence.  Successful use of chemical shift for secondary structure inference requires the correct referencing.  The LACS server gives independent CS offsets for CA, CB, CO and HA and allows for effective correction of chemical shift sets derived from [''U''-13C,15N] and from [''U''-2H,13C,15N].  The latter labeling scheme may show distinct degrees of offset for CA and CB due to the different number of bound deuterons which affect the value of the carbon to different degree.   

 

== '''Running LACS''' ==

LACS can be run on the native [http://bija.nmrfam.wisc.edu/MANI-LACS/ server] using an edited version of the chemical shift file in bmrb 2.1 format, the user's email and project name.  The server returns the results very rapidly, the chemical shift file format is stringent and the output is of straight forward interpretation. 

The items returned are:

i) a text file with the nucleus specific offsets from the linear fit of the secondary CS  (e. g. deltaCA vs. deltaCA-deltaCB), obtained by subtracting the random coil value from the atom of interest.

ii) plots of the secondary CS outliers that may reveal incorrectly assigned residues.

 

A running [[Media:WR73-ILVFY_111809_4LACS.bmrb|bmrb]] file and text [[media:LACS_output.txt|output]] example are provided for user's convenience, adhering to the format should provide smooth operation of the server. 

 

=== Example BMRB input for LACS ===
<pre> _Mol_residue_sequence
;
MLIYKDIFTDDELSSDSFPM
KLVDDLVYEFKGKHVVRKEG
EIVLAGSNPSAEEGAEDDGS
DEHVERGIDIVLNHKLVEMN
CYEDASMFKAYIKKFMKNVI
DHMEKNNRDKADVDAFKKKI
QGWVVSLLAKDRFKNLAFFI
GERAAEGAENGQVAIIEYRD
VDGTEVPTLMLVKEAIIEEK
CLE
;

loop_
_Residue_seq_code
_Residue_label
_Chem_shift_ambiguity_code
4 1 MET C C 170.742 . 1
5 1 MET CA C 54.828 . 1
6 1 MET CB C 32.579 . 1
7 2 LEU H H 8.934 . 1
8 2 LEU HD1 H 0.688 . 1

</pre>
 

=== Example LACS ouput ===

The CA correction of -0.19 ppm is found (last line).
<pre>data_LACS

#################################
# LACS Output Information #
#################################

############################################################
# LACS Designator Definition #
# #
# Index Value Definition #
# #
# 0 Outliers #
# 1 Normal points #
# #
############################################################

save_LACS_CACB_CA_output
_LACS_plot.Sf_category LACS_output
_LACS_plot.Input_file_name "data.txt"

_LACS_plot.X_coord_name CA-CB
_LACS_plot.Y_coord_name CA
_LACS_plot.Line_1_terminator_val_x_1 -6.35
_LACS_plot.Line_1_terminator_val_y_1 -2.37
_LACS_plot.Line_1_terminator_val_x_2 1.99
_LACS_plot.Line_1_terminator_val_y_2 0.93
_LACS_plot.Line_2_terminator_val_x_1 -2.00
_LACS_plot.Line_2_terminator_val_y_1 -1.18
_LACS_plot.Line_2_terminator_val_x_2 5.76
_LACS_plot.Line_2_terminator_val_y_2 4.33
_LACS_plot.Y_axis_chem_shift_offset -0.19

</pre>
 

== '''Practical Experience with Running LACS in the NESG''' ==

In the NESG the IUPAC referencing is obtained using internal 50 uM DSS and setting the DSS methyl peak to 0.00 ppm at the temperature of interest.  The spectrometer temperature is calibrated using a neat methanol sample in the 10 - 40 °C range.  Under these conditions, backbone CS of double labeled protein samples derived from triple resonance experiments alone typically show small deviations 0.1 - 0.3 ppm in 13C and 0.01 - 0.03 ppm in 1H.  The final CS set derived from double labeled samples filtered through higher resolution [1H,13C]-HSQC tends to give offsets at the lower extreme of this range.  On a smaller statistical sampling of triply labeled proteins we found higher offset values (0.4 - 0.8 ppm) in CA and CB.  Offsett correction prior to TALOS or MFR runs are therefore highly recommended especially for the treatment of triply labeled samples.  

  

 

editing of this entry is in progress (prossi) 

 

 

-- PaoloRossi - 20 Nov 2009

LACS

2009-11-20T19:18:11Z

Prossi:

== '''Introduction''' ==

Linear analysis of chemical shift ([http://www.ncbi.nlm.nih.gov/pubmed/16041479 LACS]) provides an independent means for absolute [[Www.bmrb.wisc.edu/iupac.pdf|referencing]] of protein chemical shifts.  LACS is routinely run on all chemical shift sets submitted to the BioMagResBank (BMRB). 

In the contest of protein structure determination chemical shifts (CS) play a key role as reporters of secondary structure.  Backbone chemical shift derived dihedral angles can be obtained through programs such as [http://www.ncbi.nlm.nih.gov/pubmed/19548092 TALOS+] and are used as constraints in the structure determination process. The backbone shift set (CA, CB, CO, HN, N, HA) is also used in the molecular fragment selection (MFR) for [http://www.ncbi.nlm.nih.gov/pubmed/18326625 CS-Rosetta] structure prediction protocol.  Here fragments are selected from the protein databank (PDB) that have the same predicted CS as the query sequence.  Successful use of chemical shift for secondary structure inference requires the correct referencing.  The LACS server gives independent CS offsets for CA, CB, CO and HA and allows for effective correction of chemical shift sets derived from [''U''-13C,15N] and from [''U''-2H,13C,15N].  The latter labeling scheme may show distinct degrees of offset for CA and CB due to the different number of bound deuterons which affect the value of the carbon to different degree.   

 

== '''Running LACS''' ==

LACS can be run on the native [http://bija.nmrfam.wisc.edu/MANI-LACS/ server] using an edited version of the chemical shift file in bmrb 2.1 format, the user's email and project name.  The server returns the results very rapidly, the chemical shift file format is stringent and the output is of straight forward interpretation. 

The items returned are:

i) a text file with the nucleus specific offsets from the linear fit of the secondary CS  (e. g. deltaCA vs. deltaCA-deltaCB), obtained by subtracting the random coil value from the atom of interest.

ii) plots of the secondary CS outliers that may reveal incorrectly assigned residues.

 

A running [[media:WR73-ILVFY_111809_4LACS.bmrb|bmrb]] file and text output example are provided for user's convenience, adhering to the format should provide smooth operation of the server. 

 

=== Example BMRB input for LACS ===
<pre> _Mol_residue_sequence
;
MLIYKDIFTDDELSSDSFPM
KLVDDLVYEFKGKHVVRKEG
EIVLAGSNPSAEEGAEDDGS
DEHVERGIDIVLNHKLVEMN
CYEDASMFKAYIKKFMKNVI
DHMEKNNRDKADVDAFKKKI
QGWVVSLLAKDRFKNLAFFI
GERAAEGAENGQVAIIEYRD
VDGTEVPTLMLVKEAIIEEK
CLE
;

loop_
_Residue_seq_code
_Residue_label
_Chem_shift_ambiguity_code
4 1 MET C C 170.742 . 1
5 1 MET CA C 54.828 . 1
6 1 MET CB C 32.579 . 1
7 2 LEU H H 8.934 . 1
8 2 LEU HD1 H 0.688 . 1

</pre>
 

=== Example LACS ouput ===

The CA correction of -0.19 ppm is found (last line).
<pre>data_LACS

#################################
# LACS Output Information #
#################################

############################################################
# LACS Designator Definition #
# #
# Index Value Definition #
# #
# 0 Outliers #
# 1 Normal points #
# #
############################################################

save_LACS_CACB_CA_output
_LACS_plot.Sf_category LACS_output
_LACS_plot.Input_file_name "data.txt"

_LACS_plot.X_coord_name CA-CB
_LACS_plot.Y_coord_name CA
_LACS_plot.Line_1_terminator_val_x_1 -6.35
_LACS_plot.Line_1_terminator_val_y_1 -2.37
_LACS_plot.Line_1_terminator_val_x_2 1.99
_LACS_plot.Line_1_terminator_val_y_2 0.93
_LACS_plot.Line_2_terminator_val_x_1 -2.00
_LACS_plot.Line_2_terminator_val_y_1 -1.18
_LACS_plot.Line_2_terminator_val_x_2 5.76
_LACS_plot.Line_2_terminator_val_y_2 4.33
_LACS_plot.Y_axis_chem_shift_offset -0.19

</pre>
 

== '''Practical Experience with Running LACS in the NESG''' ==

In the NESG the IUPAC referencing is obtained using internal 50 uM DSS and setting the DSS methyl peak to 0.00 ppm at the temperature of interest.  The spectrometer temperature is calibrated using a neat methanol sample in the 10 - 40 °C range.  Under these conditions, backbone CS of double labeled protein samples derived from triple resonance experiments alone typically show small deviations 0.1 - 0.3 ppm in 13C and 0.01 - 0.03 ppm in 1H.  The final CS set derived from double labeled samples filtered through higher resolution [1H,13C]-HSQC tends to give offsets at the lower extreme of this range.  On a smaller statistical sampling of triply labeled proteins we found higher offset values (0.4 - 0.8 ppm) in CA and CB.  Offsett correction prior to TALOS or MFR runs are therefore highly recommended especially for the treatment of triply labeled samples.  

  

 

editing of this entry is in progress (prossi) 

 

 

-- PaoloRossi - 20 Nov 2009

File:LACS output.txt

2009-11-20T19:16:37Z

Prossi:

2009-11-20T15:35:58Z

Prossi:

This outline of the NESG NMR Wiki is designed to expand on the existing "Master Recipe" and should serve as an experience harvesting tool.

*It has a rather broad coverage to facilitate long-tewrm growth and development. Aditional compact aggregator pages may be needed to pesent specific information concisely.
*There would be separate webs within the wiki: Public(or Main), NESG, and member lab webs. Most common knowlege topics should be public, unless they are specific to NESG
*We assume that the target audience has some knowledge about NMR and protein structure determination, but make the content useful for training
*"Resonance Assignment" and "Structure Determination" chaptes would focus on individual software packages. The XEASY resonance assignment tree, as the most complete, would serve as a template for other software.
*Most chapters should include a "general principles" section.

Please leave your comments/suggestion at the bottom of this page

 

= HTP NMR structure determination =

== Protein Target Selection, Sample Preparation, and Initial Screening ==

#[[Target selection|NESG target selection]] - overview of target selection in PSI III
#[[Bioinformatics with protein sequence]]
#[[DNA cloning protocols|DNA cloning protocols]] 
#[[Protein purification|Protein expression and purification protocols]]  
#Sample Optimization
##[[construct optimization]]
##[[Buffer optimization]]
##[[Cofactor optimization]]
#Initial protein analysis
##[[SDS page gel]]
##[[Protein concentration|Protein concentration measurements]]
##[[Oligomerization Status|Assessment of Oligomerization Status]]
###[[Gel filtration and light scattering|gel-filtration and light scattering]]
###[[Sedimentation equilibrium|Sedimentation equilibrium]]
###[[NMR determined Rotational correlation time]]
##[[MassSpectrometry|Mass spectrum]]
##NMR screening
###[[1D screening|1H 1D screening]]
###[[Nhsqc screen|Initial [15N,1H] HSQC]] 

== NMR Data Collection ==

#Routine operation
##[[NMR sample tubes]]
##[[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]]
##Custom NMR experiment setup scripts for VNMRJ
##1D 1H NMR spectra and 2D [15N, 1H]-HSQC
##[[Estimation of rotational correlation time]]
##Estimation of measurement time
##NMR experiments for spin system identification
##2D and 3D NOESY
##Double and triple NMR experiments
###3D CBCA(CO)NH and HNCACB
###3D HNCA and HN(CO)CA
###3D HAHB(CO)NH
###(4,3)D CABCA(CO)NH and HNCACB
###(4,3)D HABCAB(CO)NH
###(H)CCH
###(H)CCH-TOCSY
###H(C)CH
###H(C)CH-TOCSY
###(4,3)D HCCH
##Other NMR experiments
###2D [13C, 1H]-HSQC for 5% 13C-labeled samples
###2D [15N, 1H]-long-range-HSQC for determination of histidine protomer state
###MEXICO
###CLEANEX
###H-D exchange experiment
###15N spin relaxation parameters
#Advanced problems for data collection
##Setting up non-uniformly sampled spectra
###Guide for Varian/BioPack
###Guide for Bruker according to Arrowsmith group
#Maintenance
##VARIAN
###Installing and updating BioPack
###Full Probefile calibration
###Rebooting the console
###Cryoprobe conditioning
##BRUKER

== NMR Data Processing ==

#General Priciples and Concepts
##Fourier transformation
###Zero-filling
###Apodization
###Phasing
###Linear prediction
###G-matrix Fourier transformation (GFT)
##Alternatives to Fourier transformation
###Maximum entropy reconstruction
###MDD reconstruction
###...
#Practical Aspects
##NMRPIPE
###General information
###Buffalo's Processing Protocol using NMRpipe
##PROSA
##TOPSPIN
##[[AGNuS/AutoProc|AGNuS/AutoProc]]
##UBNMR
##Spectral format conversion

 

== Resonance Assignment ==

This chapter would focus on individual data analysis and resonance assignment packages, as most people stick to a particular software for entire structure determination projects.

 

#[[Principles and concepts]]
##[[Stable isotope labeling schemes]]
##[[NMR experiments]]
###[[Through-bond]]
###[[Through space]]
##[[Spin systems]]
###[[Definitions]]
###[[Identification]]
###[[Linking spin systems]]
###[[Matching onto covalent structure]]
#[[Practical aspects]]
##[[Semi-automated protocols]]
###[[CARA]]
####[[Spin System Identification with CARA|Spin System Identification in 2D 15N-HSQC and 3D HNNCO]]
####[[Backbone Assignment with CARA|Backbone Resonance Assignment]]
####[[HA and HB Assignment with CARA|Assignment of HA and HB Resonances with (4,3)D GFT HABCAB(CO)NHN]]
####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]]
###[[Sparky]]
###[[XEASY]]
####[[XEASY Spin system identification|Spin system identification]]
####[[XEASY Backbone Assignment|Backbone resonance assignment]]''' '''
#####GFT-based spectra
######[[HNCACAB/CABCA(CO)NH]]
#####Conventional spectra
######[[HNCACB/CBCA(CO)NH]]
######HNCA/HN(CO)CA
######HNCO/HN(CA)CO
######NOESY/TOCSY
####[[XEASY Side Chain Assignment|Side chain resonance assignment]]
#####Aliphatic
######GFT NMR spectra
#######[[HA and HB Assignment with GFT in XEASY|(4,3)D GFT HABCAB(CO)NHN]]
#######[[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|(4,3)D GFT HCCH]]
######Conventional spectra
#######HAHB(CO)NH
#######HCCH-COSY
#######HCCH-TOCSY
#######[[Side chain assignment with CN-NOESY in XEASY|Simultaneous NOESY]]
#######(H)CC-TOCSY-(CO)NH
#######H(CC-TOCSY-CO)NH
#####[[Aromatic side chain assignment with Aro-HCCH-COSY in XEASY|Aromatic]]
######GFT-based spectra
######Conventional spectra
#####Other
######Trp e1 NH and d1 CH
######[[Met methyl assignment with NOESY|Met e CH3 ]]
######[[Amide Side Chain assignment with NOESY|Asn d2 and Gln e2 NH2]]
#####NOESY peak integration
##Automated protocols
###[[AutoAssign|AutoAssign]]
###[[AutoAssign WebServer|AutoAssign server]]
###[[Abacus|ABACUS]]
###[[The PINE Server|PINE server]]
##Validation of resonance assignment
###[[AVS|AVSx]]
###[[LACS|LACSx]]
##Depositing chemical shifts

 

== Structure Calculation and Validation ==

#[[Structure Calculation and Validation|Principles and concepts]]
#Practical aspects
##Structure calculation
###CYANA
####[[CYANA|Getting started]]
####[[FOUND|FOUND]]
####[[TALOS|TALOS]]
####[[GLOMSA|GLOMSA]]
####[[NOE Calibration Using CYANA|NOE calibration]]
####[[Manual Structure Calculation Using CYANA|Manual structure calculation]]
####[[Automated NOESY Assignment Using CYANA|Automated NOESY assignment and structure calculation]]
####[[Structure Calculation With RDC's Using CYANA|Structure calculation with residual dipolar couplings]] (link to REDCAT/PALES,FINDTENSOR, .rdc file, adding ORI to PDB file)
####[[Homodimer Structure Calculation Using CYANA|Homodimer structure calculations]][[Homodimer Structure Calculation Using CYANA| ]]
###AutoStructure
####[[AutoStructure/RPF Theory|Theory]]
####[[AutoStructure|Getting started]]
####[[CYANA Structure Calculations Using AutoStructure|CYANA run]]
####[[XPLOR Structure Calculations Using AutoStructure|XPLOR run]]
####[[Analyzing AutoStructure Output Directories|Analyzing the output]]
####[[RPF Analysis|RPF/DP scores]]
####[[Structure Calculation Using AS-DP|Structure calculation using AS-DP]]
###"Consensus" Approaches
####[[Overview of Consensus Runs|Overview of Consensus runs]]
####[[Finding Consensus NOE Assignments|Finding Consensus NOE assignments]]
####[[Validation of Consensus Run|Validation of Consensus runs]]
###[[Structure Calculation Using CS-Rosetta|CS-ROSETTA]]
###[[Structure Calculation Using CS-CP ROSETTA|CS-DP ROSETTA]]
###[[Structure Calculation Using RDC-ROSETTA|RDC-ROSETTA]]
###[[RDC-Assisted Dimer Structure Determination|RDC-assisted dimer structure calculation]] 
###Special topics
####[[Protein-Ligand Complexes|Protein-Ligand complexes]]
####[[Working With Metal Ions|Metal ions]]
####[[Working With Dimers|Dimers]]
####[[Residual Dipolar Couplings in Structure Refinement|Residual Dipolar Couplings]]
####[[REDCAT|REDCAT]] and [[REDCRAFT|REDCRAFT]]
####[[Paramagnetic Constraints in Structure Determination|Paramagnetic constraints]]
##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]]

-- JeffMills - 28 May 2009

Here are two comments from Guy:

- need to have centralized site for downloading all software that NESG has developed or licensed; this would be a central site for NESG scientists to use to access the latest version of all software

- need to allow outside users to access links to all software (they will need licenses to download) and also to download software from NESG

-- AlexEletski - 13 Jul 2009

Wiki Tree Layout

2009-11-20T15:35:16Z

Prossi:

This outline of the NESG NMR Wiki is designed to expand on the existing "Master Recipe" and should serve as an experience harvesting tool.

*It has a rather broad coverage to facilitate long-tewrm growth and development. Aditional compact aggregator pages may be needed to pesent specific information concisely.
*There would be separate webs within the wiki: Public(or Main), NESG, and member lab webs. Most common knowlege topics should be public, unless they are specific to NESG
*We assume that the target audience has some knowledge about NMR and protein structure determination, but make the content useful for training
*"Resonance Assignment" and "Structure Determination" chaptes would focus on individual software packages. The XEASY resonance assignment tree, as the most complete, would serve as a template for other software.
*Most chapters should include a "general principles" section.

Please leave your comments/suggestion at the bottom of this page

 

= HTP NMR structure determination =

== Protein Target Selection, Sample Preparation, and Initial Screening ==

#[[Target selection|NESG target selection]] - overview of target selection in PSI III
#[[Bioinformatics with protein sequence]]
#[[DNA cloning protocols|DNA cloning protocols]] 
#[[Protein purification|Protein expression and purification protocols]]  
#Sample Optimization
##[[construct optimization]]
##[[Buffer optimization]]
##[[Cofactor optimization]]
#Initial protein analysis
##[[SDS page gel]]
##[[Protein concentration|Protein concentration measurements]]
##[[Oligomerization Status|Assessment of Oligomerization Status]]
###[[Gel filtration and light scattering|gel-filtration and light scattering]]
###[[Sedimentation equilibrium|Sedimentation equilibrium]]
###[[NMR determined Rotational correlation time]]
##[[MassSpectrometry|Mass spectrum]]
##NMR screening
###[[1D screening|1H 1D screening]]
###[[Nhsqc screen|Initial [15N,1H] HSQC]] 

== NMR Data Collection ==

#Routine operation
##[[NMR sample tubes]]
##[[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]]
##Custom NMR experiment setup scripts for VNMRJ
##1D 1H NMR spectra and 2D [15N, 1H]-HSQC
##[[Estimation of rotational correlation time]]
##Estimation of measurement time
##NMR experiments for spin system identification
##2D and 3D NOESY
##Double and triple NMR experiments
###3D CBCA(CO)NH and HNCACB
###3D HNCA and HN(CO)CA
###3D HAHB(CO)NH
###(4,3)D CABCA(CO)NH and HNCACB
###(4,3)D HABCAB(CO)NH
###(H)CCH
###(H)CCH-TOCSY
###H(C)CH
###H(C)CH-TOCSY
###(4,3)D HCCH
##Other NMR experiments
###2D [13C, 1H]-HSQC for 5% 13C-labeled samples
###2D [15N, 1H]-long-range-HSQC for determination of histidine protomer state
###MEXICO
###CLEANEX
###H-D exchange experiment
###15N spin relaxation parameters
#Advanced problems for data collection
##Setting up non-uniformly sampled spectra
###Guide for Varian/BioPack
###Guide for Bruker according to Arrowsmith group
#Maintenance
##VARIAN
###Installing and updating BioPack
###Full Probefile calibration
###Rebooting the console
###Cryoprobe conditioning
##BRUKER

== NMR Data Processing ==

#General Priciples and Concepts
##Fourier transformation
###Zero-filling
###Apodization
###Phasing
###Linear prediction
###G-matrix Fourier transformation (GFT)
##Alternatives to Fourier transformation
###Maximum entropy reconstruction
###MDD reconstruction
###...
#Practical Aspects
##NMRPIPE
###General information
###Buffalo's Processing Protocol using NMRpipe
##PROSA
##TOPSPIN
##[[AGNuS/AutoProc|AGNuS/AutoProc]]
##UBNMR
##Spectral format conversion

 

== Resonance Assignment ==

This chapter would focus on individual data analysis and resonance assignment packages, as most people stick to a particular software for entire structure determination projects.

 

#[[Principles and concepts]]
##[[Stable isotope labeling schemes]]
##[[NMR experiments]]
###[[Through-bond]]
###[[Through space]]
##[[Spin systems]]
###[[Definitions]]
###[[Identification]]
###[[Linking spin systems]]
###[[Matching onto covalent structure]]
#[[Practical aspects]]
##[[Semi-automated protocols]]
###[[CARA]]
####[[Spin System Identification with CARA|Spin System Identification in 2D 15N-HSQC and 3D HNNCO]]
####[[Backbone Assignment with CARA|Backbone Resonance Assignment]]
####[[HA and HB Assignment with CARA|Assignment of HA and HB Resonances with (4,3)D GFT HABCAB(CO)NHN]]
####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]]
###[[Sparky]]
###[[XEASY]]
####[[XEASY Spin system identification|Spin system identification]]
####[[XEASY Backbone Assignment|Backbone resonance assignment]]''' '''
#####GFT-based spectra
######[[HNCACAB/CABCA(CO)NH]]
#####Conventional spectra
######[[HNCACB/CBCA(CO)NH]]
######HNCA/HN(CO)CA
######HNCO/HN(CA)CO
######NOESY/TOCSY
####[[XEASY Side Chain Assignment|Side chain resonance assignment]]
#####Aliphatic
######GFT NMR spectra
#######[[HA and HB Assignment with GFT in XEASY|(4,3)D GFT HABCAB(CO)NHN]]
#######[[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|(4,3)D GFT HCCH]]
######Conventional spectra
#######HAHB(CO)NH
#######HCCH-COSY
#######HCCH-TOCSY
#######[[Side chain assignment with CN-NOESY in XEASY|Simultaneous NOESY]]
#######(H)CC-TOCSY-(CO)NH
#######H(CC-TOCSY-CO)NH
#####[[Aromatic side chain assignment with Aro-HCCH-COSY in XEASY|Aromatic]]
######GFT-based spectra
######Conventional spectra
#####Other
######Trp e1 NH and d1 CH
######[[Met methyl assignment with NOESY|Met e CH3 ]]
######[[Amide Side Chain assignment with NOESY|Asn d2 and Gln e2 NH2]]
#####NOESY peak integration
##Automated protocols
###[[AutoAssign|AutoAssign]]
###[[AutoAssign WebServer|AutoAssign server]]
###[[Abacus|ABACUS]]
###[[The PINE Server|PINE server]]
##Validation of resonance assignment
###[[AVS|AVS]]
###[[LACS|LACS]]
##Depositing chemical shifts

 

== Structure Calculation and Validation ==

#[[Structure Calculation and Validation|Principles and concepts]]
#Practical aspects
##Structure calculation
###CYANA
####[[CYANA|Getting started]]
####[[FOUND|FOUND]]
####[[TALOS|TALOS]]
####[[GLOMSA|GLOMSA]]
####[[NOE Calibration Using CYANA|NOE calibration]]
####[[Manual Structure Calculation Using CYANA|Manual structure calculation]]
####[[Automated NOESY Assignment Using CYANA|Automated NOESY assignment and structure calculation]]
####[[Structure Calculation With RDC's Using CYANA|Structure calculation with residual dipolar couplings]] (link to REDCAT/PALES,FINDTENSOR, .rdc file, adding ORI to PDB file)
####[[Homodimer Structure Calculation Using CYANA|Homodimer structure calculations]][[Homodimer Structure Calculation Using CYANA| ]]
###AutoStructure
####[[AutoStructure/RPF Theory|Theory]]
####[[AutoStructure|Getting started]]
####[[CYANA Structure Calculations Using AutoStructure|CYANA run]]
####[[XPLOR Structure Calculations Using AutoStructure|XPLOR run]]
####[[Analyzing AutoStructure Output Directories|Analyzing the output]]
####[[RPF Analysis|RPF/DP scores]]
####[[Structure Calculation Using AS-DP|Structure calculation using AS-DP]]
###"Consensus" Approaches
####[[Overview of Consensus Runs|Overview of Consensus runs]]
####[[Finding Consensus NOE Assignments|Finding Consensus NOE assignments]]
####[[Validation of Consensus Run|Validation of Consensus runs]]
###[[Structure Calculation Using CS-Rosetta|CS-ROSETTA]]
###[[Structure Calculation Using CS-CP ROSETTA|CS-DP ROSETTA]]
###[[Structure Calculation Using RDC-ROSETTA|RDC-ROSETTA]]
###[[RDC-Assisted Dimer Structure Determination|RDC-assisted dimer structure calculation]] 
###Special topics
####[[Protein-Ligand Complexes|Protein-Ligand complexes]]
####[[Working With Metal Ions|Metal ions]]
####[[Working With Dimers|Dimers]]
####[[Residual Dipolar Couplings in Structure Refinement|Residual Dipolar Couplings]]
####[[REDCAT|REDCAT]] and [[REDCRAFT|REDCRAFT]]
####[[Paramagnetic Constraints in Structure Determination|Paramagnetic constraints]]
##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]]

-- JeffMills - 28 May 2009

Here are two comments from Guy:

- need to have centralized site for downloading all software that NESG has developed or licensed; this would be a central site for NESG scientists to use to access the latest version of all software

- need to allow outside users to access links to all software (they will need licenses to download) and also to download software from NESG

-- AlexEletski - 13 Jul 2009