NESG Wiki - User contributions [en]

Residual Dipolar Couplings in Structure Refinement

2012-05-11T17:07:35Z

Lmorris:

== '''Introduction''' ==

Residual Dipolar Couplings (RDCs) originate from partial anisotropic averaging of the dipolar interaction which is dependent on the angle between an internuclear vector and the magnetic field. When a molecule sample orientations uniformly, as it does in normal solution NMR, RDCs average to zero and are not observable. However, if a molecule is dissolved in a dilute liquid crystalline medium it becomes partially aligned, and the dipolar couplings are not completely averaged to zero, leading to a small contribution to the splittings of NMR signals. The angular dependence of these contributions can provide valuable structural information. Protein structures can be validated using RDCs, and structures can be refined to improve quality.

Another application of RDCs is identification of the correct monomer orientation in homodimers. This application requires RDCs from two or more alignment media that give different orientations of principal alignment axes.

Below are protocols for the refinement of protein structures with RDCs using XPLOR-NIH [1].

 

== '''RDC Refinement Using XPLOR-NIH''' ==

=== Using XPLOR-NIH ===

X-PLOR can be downloaded from [http://nmr.cit.nih.gov/xplor-nih/ http://nmr.cit.nih.gov/xplor-nih/]. 

 

*Obtain a good estimate of the magnitude of Da and R from the alignment tensors. The alignment tensors can be obtained either from the extremes of distribution of RDCs (which forms a histogram) or from the program REDCAT. REDCAT gives values for Sxx, Syy and Szz. These values are relateive to the maxium RDC (RDCmax) for that bond type. First, multiply these values from REDCAT by the associated maxium RDC value (for example 24350 for NH dipolar couplings). These alignment parameters are related to Da and R as follows:
<pre>Da = RDCmax * Szz/2
R = Dr/Da where Dr = 0.5 * (Sxx-Syy) * RDCmax
</pre>
*Note that the sani constraint in XPLOR and or CNS requires three coefficients: DFS, Da and R. DFS is a fixed offset. For RDC data, DFS = 0.
*In XPLOR define the axis representing the alignment tensor coordinate system. The coordinate system is represented by four pseudo atoms: OO(origin), X ,Y and Z. The coordinate system has to be positioned far away to prevent any interaction with the protein. For more than one aligned media, define separate axes for each medium. These axes can either be defined at the end of the pdb file or as a separate .pdb file. Shown below is an example from the pdb:
<pre>ATOM 608 X ANI 500 33.000 30.000 30.000 1.00 0.00
ATOM 609 Y ANI 500 30.000 33.000 30.000 1.00 0.00
ATOM 610 Z ANI 500 30.000 30.000 33.000 1.00 0.00
ATOM 611 OO ANI 500 30.000 30.000 30.000 1.00 0.00
END
</pre>
You can also create a separate, standalone pdb file ('axis.pdb') with only one residue starting with ATOM 1. This separate pdb file can then be read in XPLOR or CNS as an additional coordinate file using the <tt>coor</tt> command.

 

*Create an RDC constraint file. An example is shown below where -8.1 is the coupling value and plus and minus 0.5 is the error associated with it:
<pre>assign ( resid 500 and name OO )
( resid 500 and name Z )
( resid 500 and name X )
( resid 500 and name Y )
( resid 1 and name HN )
( resid 1 and name N ) -8.1 0.5 0.5
</pre>
 

*The following perl script ('makeRdcAssign.pm') can be used to create an RDC constraint file ('myprotRdc.tbl'). The input to the script is coupling values as one column text file (same as used in REDCAT to import rdcs). Usage for this script is: <tt>makeRdcAssign.pm myprot.rdc myprotRdc.tbl startingResidue Err1 Err2</tt>. The same script can be used for CNS, the only change is to make a tbl file with only one error Err1.
<pre>#!/usr/bin/perl
if($ARGC < 3 ) {
print "Usage: makeRDCAssign.pm infile outfile startingResidue# <err1> <err2> \n";
}
$fileIn = $ARGV[0];
$fileOut = $ARGV[1];
$startRes = $ARGV[2];
$rdcE1 = 0.6;
$rdcE2 = 0.5;
$useE2 = 0;

if ($ARGV[3] ){
$rdcE1 = $ARGV[3];
}
if ($ARGV[4] ){
$rdcE2 = $ARGV[4];
$useE2 = 1;
}
unless( open(RDC_IN, $fileIn)) {
die("could not open input file $fileIn \n");
}
unless( open(RDC_OUT, ">$fileOut")) {
die("could not open outpu file $fileOut \n");
}

$thisRes = $startRes;
while ($thisLine = <RDC_IN> ) {
@parts = split(/\s+/,$thisLine ); # split by spaces
$thisRDC = $parts[0];
print("residue $thisRes, rdc $thisRDC \n");

# if the rdc is valid, write to the tbl file
if ($thisRDC < 999 ) {
print RDC_OUT "assign ( resid 500 and name OO ) \n";
print RDC_OUT " ( resid 500 and name Z ) \n";
print RDC_OUT " ( resid 500 and name X ) \n";
print RDC_OUT " ( resid 500 and name Y ) \n";
print RDC_OUT " ( resid $thisRes and name HN ) \n";
print RDC_OUT " ( resid $thisRes and name N ) ";
if ($useE2 > 0 ) {
print RDC_OUT " $thisRDC $rdcE1 $rdcE2 \n\n";
} else {
print RDC_OUT " $thisRDC $rdcE1 \n\n";
}
}
$thisRes = $thisRes + 1;
}
</pre>
 

*Do not include all the RDCs for refinement. Leave 15-20% of the RDCs for validation. 

*Set the Xplor-NIH input file for dipolar coupling refinement. The RDC restraint is known as sani (susceptibility anisotropy). Define the force constant, ksani for each media and for each type of couplings. 

*The refined structure can be validated with the RDCs that are not used in refinement. From the refined structure RDCs can be back calculated (using REDCAT) and plotted against the experimental RDCs. 

 

=== Details for performing RDC refinement in XPLOR/NIH starting from CNS ===

*Prepare the RDC constraint file as discussed above.
*Prepare noe and dihedral angle constraint files as for CNS ("aco.tbl", noe.tbl")
*The nomenclature and thus the topology of methyl groups in XPLOR is NOT the same as for CNS. You must rebuild each of your CNS pdb files into XPLOR format using the script <tt>generate1.inp</tt>. Edit the script and replace <tt>myprot</tt> with the appropriate prefix for your protein. generate.inp will also generate a structure file ("myprot.psf") for use with XPLOR. 
*The script "doi" will repeatedly run generate1.inp, iterating over all of your input structures.
*NOTE: generate1.inp does not like atoms OT1 and OT2 on the C terminal. Get rid of one and replace the other with O, make sure you preserve the exact column alignment in the pdb file.
*Refinment with RDCs sets anglular constraints relative to a virtual "axis" residue. You must also have a copy of the axis residue pdb file ("axis.pdb") in the local directory.
*The script refine_rdc_myprot.inp can be used to run the refinement including RDC constraints.
*You must edit the script to provide the correct names for your noe, dihedral and rdc input files xxx.tbl, you must also edit the coefficients for the NH sani constraints to put in your Da and Rhombicity as calculated above.
*The script "dos" will repeatedly run refine_rdc_myprot.inp, iterating over all of your input structures and creating refined structures.
*These refined structures may then be directly used in CNS if water bath refinement is desired.

 

=== Details for performing RDC refinement in XPLOR/NIH using Python Interface ===

XPLOR-NIH can be used by python interface. It does offer many conveniences over the old XPLOR script interface, for instance, no longer there is need to setup the .psf file for the alignment tensor and the value of Da and R don't have to be fixed. The details for the usage can be found on the tutorial attached. With the new version of XPLOR-NIH refine.py script can be found in the eginput directory. The script can be modified based on the number of media, values of Da and R. Below is the sani part of the script for the refrence. 
<pre># orientation Tensor - used with the dipolar coupling term
# one for each medium
# For each medium, specify a name, and initial values of Da, Rh.
#
from varTensorTools import create_VarTensor
media={}
# medium Da rhombicity
for (medium,Da,Rh) in [ ('t', -6.5, 0.62),
('b', -9.9, 0.23) ]:
oTensor = create_VarTensor(medium)
oTensor.setDa(Da)
oTensor.setRh(Rh)
media[medium] = oTensor
pass
# dipolar coupling restraints for protein amide NH.
# collect all RDCs in the rdcs PotList
# RDC scaling. Three possible contributions.
# 1) gamma_A * gamma_B / r_AB^3 prefactor. So that the same Da can be used
# for different expts. in the same medium. Sometimes the data is
# prescaled so that this is not needed. scale_toNH() is used for this.
# Note that if the expt. data has been prescaled, the values for rdc rmsd
# reported in the output will relative to the scaled values- not the expt.
# values.
# 2) expt. error scaling. Used here. A scale factor equal to 1/err^2
# (relative to that for NH) is used.
# 3) sometimes the reciprocal of the Da^2 is used if there is a large
# spread in Da values. Not used here.
#
from rdcPotTools import create_RDCPot, scale_toNH
rdcs = PotList('rdc')
for (medium,expt,file, scale) in \
[('t','NH' ,'tmv107_nh.tbl' ,1),
('t','NCO','tmv107_nc.tbl' ,.05),
('t','HNC','tmv107_hnc.tbl' ,.108),
('b','NH' ,'bicelles_new_nh.tbl' ,1),
('b','NCO','bicelles_new_nc.tbl' ,.05),
('b','HNC','bicelles_new_hnc.tbl',.108)
]:
rdc = create_RDCPot("%s_%s"%(medium,expt),file,media[medium])

#1) scale prefactor relative to NH
# see python/rdcPotTools.py for exact calculation
# scale_toNH(rdc) - not needed for these datasets -
# but non-NH reported rmsd values will be wrong.

#3) Da rescaling factor (separate multiplicative factor)
# scale *= ( 9.9 / rdcs[name].oTensor.Da(0) )**2
rdc.setScale(scale)
rdc.setShowAllRestraints(1) #all restraints are printed during analysis
rdc.setThreshold(1.5) # in Hz
rdcs.append(rdc)
pass
potList.append(rdcs)
rampedParams.append( MultRamp(0.05,5.0, "rdcs.setScale( VALUE )") )
</pre>
The command to run python script is
<pre>xplor -py refine.py </pre>
== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/12565051?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=3 1.    Schwieters, C.D., Kuszewski, J.J., Tjandra, N. and Clore, G.M.  (2003) The Xplor-NIH NMR molecular structure determination package.  ''J. Magn. Res. 160'', 65-73.]

Residual Dipolar Couplings in Structure Refinement

2012-05-11T17:06:51Z

Lmorris:

== '''Introduction''' ==

Residual Dipolar Couplings (RDCs) originate from partial anisotropic averaging of the dipolar interaction which is dependent on the angle between an internuclear vector and the magnetic field. When a molecule sample orientations uniformly, as it does in normal solution NMR, RDCs average to zero and are not observable. However, if a molecule is dissolved in a dilute liquid crystalline medium it becomes partially aligned, and the dipolar couplings are not completely averaged to zero, leading to a small contribution to the splittings of NMR signals. The angular dependence of these contributions can provide valuable structural information. Protein structures can be validated using RDCs, and structures can be refined to improve quality.

Another application of RDCs is identification of the correct monomer orientation in homodimers. This application requires RDCs from two or more alignment media that give different orientations of principal alignment axes.

Below are protocols for the refinement of protein structures with RDCs using XPLOR-NIH [1].

 

== '''RDC Refinement Using XPLOR-NIH''' ==

=== Using XPLOR-NIH ===

X-PLOR can be downloaded from [http://nmr.cit.nih.gov/xplor-nih/ http://nmr.cit.nih.gov/xplor-nih/]. 

 

*Obtain a good estimate of the magnitude of Da and R from the alignment tensors. The alignment tensors can be obtained either from the extremes of distribution of RDCs (which forms a histogram) or from the program REDCAT. REDCAT gives values for Sxx, Syy and Szz. These values are relateive to the maxium RDC (RDCmax) for that bond type. First, multiply these values from REDCAT by the associated maxium RDC value (for example 24350 for NH dipolar couplings). These alignment parameters are related to Da and R as follows:
<pre>Da = RDCmax * Szz/2
R = Dr/Da where Dr = 0.5 * (Sxx-Syy) * RDCmax
</pre>
*Note that the sani constraint in XPLOR and or CNS requires three coefficients: DFS, Da and R. DFS is a fixed offset. For RDC data, DFS = 0.
*In XPLOR define the axis representing the alignment tensor coordinate system. The coordinate system is represented by four pseudo atoms: OO(origin), X ,Y and Z. The coordinate system has to be positioned far away to prevent any interaction with the protein. For more than one aligned media, define separate axes for each medium. These axes can either be defined at the end of the pdb file or as a separate .pdb file. Shown below is an example from the pdb:
<pre>ATOM 608 X ANI 500 33.000 30.000 30.000 1.00 0.00
ATOM 609 Y ANI 500 30.000 33.000 30.000 1.00 0.00
ATOM 610 Z ANI 500 30.000 30.000 33.000 1.00 0.00
ATOM 611 OO ANI 500 30.000 30.000 30.000 1.00 0.00
END
</pre>
You can also create a separate, standalone pdb file ('axis.pdb') with only one residue starting with ATOM 1. This separate pdb file can then be read in XPLOR or CNS as an additional coordinate file using the <tt>coor</tt> command.

 

*Create an RDC constraint file. An example is shown below where -8.1 is the coupling value and plus and minus 0.5 is the error associated with it:
<pre>assign ( resid 500 and name OO )
( resid 500 and name Z )
( resid 500 and name X )
( resid 500 and name Y )
( resid 1 and name HN )
( resid 1 and name N ) -8.1 0.6 0.5
</pre>
 

*The following perl script ('makeRdcAssign.pm') can be used to create an RDC constraint file ('myprotRdc.tbl'). The input to the script is coupling values as one column text file (same as used in REDCAT to import rdcs). Usage for this script is: <tt>makeRdcAssign.pm myprot.rdc myprotRdc.tbl startingResidue Err1 Err2</tt>. The same script can be used for CNS, the only change is to make a tbl file with only one error Err1.
<pre>#!/usr/bin/perl
if($ARGC < 3 ) {
print "Usage: makeRDCAssign.pm infile outfile startingResidue# <err1> <err2> \n";
}
$fileIn = $ARGV[0];
$fileOut = $ARGV[1];
$startRes = $ARGV[2];
$rdcE1 = 0.6;
$rdcE2 = 0.5;
$useE2 = 0;

if ($ARGV[3] ){
$rdcE1 = $ARGV[3];
}
if ($ARGV[4] ){
$rdcE2 = $ARGV[4];
$useE2 = 1;
}
unless( open(RDC_IN, $fileIn)) {
die("could not open input file $fileIn \n");
}
unless( open(RDC_OUT, ">$fileOut")) {
die("could not open outpu file $fileOut \n");
}

$thisRes = $startRes;
while ($thisLine = <RDC_IN> ) {
@parts = split(/\s+/,$thisLine ); # split by spaces
$thisRDC = $parts[0];
print("residue $thisRes, rdc $thisRDC \n");

# if the rdc is valid, write to the tbl file
if ($thisRDC < 999 ) {
print RDC_OUT "assign ( resid 500 and name OO ) \n";
print RDC_OUT " ( resid 500 and name Z ) \n";
print RDC_OUT " ( resid 500 and name X ) \n";
print RDC_OUT " ( resid 500 and name Y ) \n";
print RDC_OUT " ( resid $thisRes and name HN ) \n";
print RDC_OUT " ( resid $thisRes and name N ) ";
if ($useE2 > 0 ) {
print RDC_OUT " $thisRDC $rdcE1 $rdcE2 \n\n";
} else {
print RDC_OUT " $thisRDC $rdcE1 \n\n";
}
}
$thisRes = $thisRes + 1;
}
</pre>
 

*Do not include all the RDCs for refinement. Leave 15-20% of the RDCs for validation. 

*Set the Xplor-NIH input file for dipolar coupling refinement. The RDC restraint is known as sani (susceptibility anisotropy). Define the force constant, ksani for each media and for each type of couplings. 

*The refined structure can be validated with the RDCs that are not used in refinement. From the refined structure RDCs can be back calculated (using REDCAT) and plotted against the experimental RDCs. 

 

=== Details for performing RDC refinement in XPLOR/NIH starting from CNS ===

*Prepare the RDC constraint file as discussed above.
*Prepare noe and dihedral angle constraint files as for CNS ("aco.tbl", noe.tbl")
*The nomenclature and thus the topology of methyl groups in XPLOR is NOT the same as for CNS. You must rebuild each of your CNS pdb files into XPLOR format using the script <tt>generate1.inp</tt>. Edit the script and replace <tt>myprot</tt> with the appropriate prefix for your protein. generate.inp will also generate a structure file ("myprot.psf") for use with XPLOR. 
*The script "doi" will repeatedly run generate1.inp, iterating over all of your input structures.
*NOTE: generate1.inp does not like atoms OT1 and OT2 on the C terminal. Get rid of one and replace the other with O, make sure you preserve the exact column alignment in the pdb file.
*Refinment with RDCs sets anglular constraints relative to a virtual "axis" residue. You must also have a copy of the axis residue pdb file ("axis.pdb") in the local directory.
*The script refine_rdc_myprot.inp can be used to run the refinement including RDC constraints.
*You must edit the script to provide the correct names for your noe, dihedral and rdc input files xxx.tbl, you must also edit the coefficients for the NH sani constraints to put in your Da and Rhombicity as calculated above.
*The script "dos" will repeatedly run refine_rdc_myprot.inp, iterating over all of your input structures and creating refined structures.
*These refined structures may then be directly used in CNS if water bath refinement is desired.

 

=== Details for performing RDC refinement in XPLOR/NIH using Python Interface ===

XPLOR-NIH can be used by python interface. It does offer many conveniences over the old XPLOR script interface, for instance, no longer there is need to setup the .psf file for the alignment tensor and the value of Da and R don't have to be fixed. The details for the usage can be found on the tutorial attached. With the new version of XPLOR-NIH refine.py script can be found in the eginput directory. The script can be modified based on the number of media, values of Da and R. Below is the sani part of the script for the refrence. 
<pre># orientation Tensor - used with the dipolar coupling term
# one for each medium
# For each medium, specify a name, and initial values of Da, Rh.
#
from varTensorTools import create_VarTensor
media={}
# medium Da rhombicity
for (medium,Da,Rh) in [ ('t', -6.5, 0.62),
('b', -9.9, 0.23) ]:
oTensor = create_VarTensor(medium)
oTensor.setDa(Da)
oTensor.setRh(Rh)
media[medium] = oTensor
pass
# dipolar coupling restraints for protein amide NH.
# collect all RDCs in the rdcs PotList
# RDC scaling. Three possible contributions.
# 1) gamma_A * gamma_B / r_AB^3 prefactor. So that the same Da can be used
# for different expts. in the same medium. Sometimes the data is
# prescaled so that this is not needed. scale_toNH() is used for this.
# Note that if the expt. data has been prescaled, the values for rdc rmsd
# reported in the output will relative to the scaled values- not the expt.
# values.
# 2) expt. error scaling. Used here. A scale factor equal to 1/err^2
# (relative to that for NH) is used.
# 3) sometimes the reciprocal of the Da^2 is used if there is a large
# spread in Da values. Not used here.
#
from rdcPotTools import create_RDCPot, scale_toNH
rdcs = PotList('rdc')
for (medium,expt,file, scale) in \
[('t','NH' ,'tmv107_nh.tbl' ,1),
('t','NCO','tmv107_nc.tbl' ,.05),
('t','HNC','tmv107_hnc.tbl' ,.108),
('b','NH' ,'bicelles_new_nh.tbl' ,1),
('b','NCO','bicelles_new_nc.tbl' ,.05),
('b','HNC','bicelles_new_hnc.tbl',.108)
]:
rdc = create_RDCPot("%s_%s"%(medium,expt),file,media[medium])

#1) scale prefactor relative to NH
# see python/rdcPotTools.py for exact calculation
# scale_toNH(rdc) - not needed for these datasets -
# but non-NH reported rmsd values will be wrong.

#3) Da rescaling factor (separate multiplicative factor)
# scale *= ( 9.9 / rdcs[name].oTensor.Da(0) )**2
rdc.setScale(scale)
rdc.setShowAllRestraints(1) #all restraints are printed during analysis
rdc.setThreshold(1.5) # in Hz
rdcs.append(rdc)
pass
potList.append(rdcs)
rampedParams.append( MultRamp(0.05,5.0, "rdcs.setScale( VALUE )") )
</pre>
The command to run python script is
<pre>xplor -py refine.py </pre>
== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/12565051?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=3 1.    Schwieters, C.D., Kuszewski, J.J., Tjandra, N. and Clore, G.M.  (2003) The Xplor-NIH NMR molecular structure determination package.  ''J. Magn. Res. 160'', 65-73.]

Residual Dipolar Couplings in Structure Refinement

2012-05-11T17:01:45Z

Lmorris:

== '''Introduction''' ==

Residual Dipolar Couplings (RDCs) originate from partial anisotropic averaging of the dipolar interaction which is dependent on the angle between an internuclear vector and the magnetic field. When a molecule sample orientations uniformly, as it does in normal solution NMR, RDCs average to zero and are not observable. However, if a molecule is dissolved in a dilute liquid crystalline medium it becomes partially aligned, and the dipolar couplings are not completely averaged to zero, leading to a small contribution to the splittings of NMR signals. The angular dependence of these contributions can provide valuable structural information. Protein structures can be validated using RDCs, and structures can be refined to improve quality.

Another application of RDCs is identification of the correct monomer orientations in homodimers. This application requires RDCs from two or more alignment media that give different orientations of principal alignment axes.

Below are protocols for the refinement of protein structures with RDCs using XPLOR-NIH [1].

 

== '''RDC Refinement Using XPLOR-NIH''' ==

=== Using XPLOR-NIH ===

X-PLOR can be downloaded from [http://nmr.cit.nih.gov/xplor-nih/ http://nmr.cit.nih.gov/xplor-nih/]. 

 

*Obtain a good estimate of the magnitude of Da and R from the alignment tensors. The alignment tensors can be obtained either from the extremes of distribution of RDCs (which forms a histogram) or from “REDCAT”. REDCAT gives values for Sxx, Syy and Szz. These values are relateive to the maxium RDC for that bond type. First, multiply these values from REDCAT by the associated maxium RDC value (for example 24350 for NH dipolar couplings). These absolute alignment tensors are related to Da and R as follows:
<pre>Da = Szz/2
R=Dr/Da where Dr=0.5*(Sxx-Syy)
</pre>
*Note that the sani constraint in XPLOR and or CNS requires three coefficients: DFS, Da and R. DFS is a fixed offset (for example a scalar coupling). For RDC data, DFS = 0.
*In XPLOR define the axis representing the alignment tensor coordinate system. The coordinate system is represented by four pseudo atoms: OO(origin), X ,Y and Z. The coordinate system has to be positioned far away to prevent any interaction with the protein. For more than one aligned media, define separate axes for each medium. These axes can either be defined at the end of the pdb file or as a separate .pdb file. Shown below is an example from the pdb:
<pre>ATOM 608 X ANI 500 33.000 30.000 30.000 1.00 0.00
ATOM 609 Y ANI 500 30.000 33.000 30.000 1.00 0.00
ATOM 610 Z ANI 500 30.000 30.000 33.000 1.00 0.00
ATOM 611 OO ANI 500 30.000 30.000 30.000 1.00 0.00
END
</pre>
You can also create a separate, standalone pdb file ('axis.pdb') with only one residue starting with ATOM 1. This separate pdb file can then be read in XPLOR or CNS as an additional coordinate file using the <tt>coor</tt> command.

 

*Create an RDC constraint file. An example is shown below where -8.1 is the coupling value and 0.6 and 0.5 are the errors associated with it:
<pre>assign ( resid 500 and name OO )
( resid 500 and name Z )
( resid 500 and name X )
( resid 500 and name Y )
( resid 1 and name HN )
( resid 1 and name N ) -8.1 0.6 0.5
</pre>
 

*The following perl script ('makeRdcAssign.pm') can be used to create RDC constraint file ('myprotRdc.tbl'). The input to the script is coupling values as one column text file (same as used in REDCAT to import rdcs). Usage for this script is <tt>makeRdcAssign.pm myprot.rdc myprotRdc.tbl startingResidue Err1 Err2</tt>. Same script can be used for CNS, the only change is to make a tbl file with only one error Err1.
<pre>#!/usr/bin/perl
if($ARGC < 3 ) {
print "Usage: makeRDCAssign.pm infile outfile startingResidue# <err1> <err2> \n";
}
$fileIn = $ARGV[0];
$fileOut = $ARGV[1];
$startRes = $ARGV[2];
$rdcE1 = 0.6;
$rdcE2 = 0.5;
$useE2 = 0;

if ($ARGV[3] ){
$rdcE1 = $ARGV[3];
}
if ($ARGV[4] ){
$rdcE2 = $ARGV[4];
$useE2 = 1;
}
unless( open(RDC_IN, $fileIn)) {
die("could not open input file $fileIn \n");
}
unless( open(RDC_OUT, ">$fileOut")) {
die("could not open outpu file $fileOut \n");
}

$thisRes = $startRes;
while ($thisLine = <RDC_IN> ) {
@parts = split(/\s+/,$thisLine ); # split by spaces
$thisRDC = $parts[0];
print("residue $thisRes, rdc $thisRDC \n");

# if the rdc is valid, write to the tbl file
if ($thisRDC < 999 ) {
print RDC_OUT "assign ( resid 500 and name OO ) \n";
print RDC_OUT " ( resid 500 and name Z ) \n";
print RDC_OUT " ( resid 500 and name X ) \n";
print RDC_OUT " ( resid 500 and name Y ) \n";
print RDC_OUT " ( resid $thisRes and name HN ) \n";
print RDC_OUT " ( resid $thisRes and name N ) ";
if ($useE2 > 0 ) {
print RDC_OUT " $thisRDC $rdcE1 $rdcE2 \n\n";
} else {
print RDC_OUT " $thisRDC $rdcE1 \n\n";
}
}
$thisRes = $thisRes + 1;
}
</pre>
 

*Do not include all the RDCs for refinement. Leave 15-20% of the RDCs for validation. 

*Set the Xplor-NIH input file for dipolar coupling refinement. The RDC restraint is known as sani (susceptibility anisotropy). Define the force constant, ksani for each media and for each type of couplings. 

*The refined structure can be validated with the RDCs that are not used in refinement. From the refined structure RDCs can be back calculated (using REDCAT) and plotted against the experimental RDCs. 

 

=== Details for performing RDC refinement in XPLOR/NIH starting from CNS ===

*Prepare the RDC constraint file as discussed above.
*Prepare noe and dihedral angle constraint files as for CNS ("aco.tbl", noe.tbl")
*The nomenclature and thus the topology of methyl groups in XPLOR is NOT the same as for CNS. You must rebuild each of your CNS pdb files into XPLOR format using the script <tt>generate1.inp</tt>. Edit the script and replace <tt>myprot</tt> with the appropriate prefix for your protein. generate.inp will also generate a structure file ("myprot.psf") for use with XPLOR. 
*The script "doi" will repeatedly run generate1.inp, iterating over all of your input structures.
*NOTE: generate1.inp does not like atoms OT1 and OT2 on the C terminal. Get rid of one and replace the other with O, make sure you preserve the exact column alignment in the pdb file.
*Refinment with RDCs sets anglular constraints relative to a virtual "axis" residue. You must also have a copy of the axis residue pdb file ("axis.pdb") in the local directory.
*The script refine_rdc_myprot.inp can be used to run the refinement including RDC constraints.
*You must edit the script to provide the correct names for your noe, dihedral and rdc input files xxx.tbl, you must also edit the coefficients for the NH sani constraints to put in your Da and Rhombicity as calculated above.
*The script "dos" will repeatedly run refine_rdc_myprot.inp, iterating over all of your input structures and creating refined structures.
*These refined structures may then be directly used in CNS if water bath refinement is desired.

 

=== Details for performing RDC refinement in XPLOR/NIH using Python Interface ===

XPLOR-NIH can be used by python interface. It does offer many conveniences over the old XPLOR script interface, for instance, no longer there is need to setup the .psf file for the alignment tensor and the value of Da and R don't have to be fixed. The details for the usage can be found on the tutorial attached. With the new version of XPLOR-NIH refine.py script can be found in the eginput directory. The script can be modified based on the number of media, values of Da and R. Below is the sani part of the script for the refrence. 
<pre># orientation Tensor - used with the dipolar coupling term
# one for each medium
# For each medium, specify a name, and initial values of Da, Rh.
#
from varTensorTools import create_VarTensor
media={}
# medium Da rhombicity
for (medium,Da,Rh) in [ ('t', -6.5, 0.62),
('b', -9.9, 0.23) ]:
oTensor = create_VarTensor(medium)
oTensor.setDa(Da)
oTensor.setRh(Rh)
media[medium] = oTensor
pass
# dipolar coupling restraints for protein amide NH.
# collect all RDCs in the rdcs PotList
# RDC scaling. Three possible contributions.
# 1) gamma_A * gamma_B / r_AB^3 prefactor. So that the same Da can be used
# for different expts. in the same medium. Sometimes the data is
# prescaled so that this is not needed. scale_toNH() is used for this.
# Note that if the expt. data has been prescaled, the values for rdc rmsd
# reported in the output will relative to the scaled values- not the expt.
# values.
# 2) expt. error scaling. Used here. A scale factor equal to 1/err^2
# (relative to that for NH) is used.
# 3) sometimes the reciprocal of the Da^2 is used if there is a large
# spread in Da values. Not used here.
#
from rdcPotTools import create_RDCPot, scale_toNH
rdcs = PotList('rdc')
for (medium,expt,file, scale) in \
[('t','NH' ,'tmv107_nh.tbl' ,1),
('t','NCO','tmv107_nc.tbl' ,.05),
('t','HNC','tmv107_hnc.tbl' ,.108),
('b','NH' ,'bicelles_new_nh.tbl' ,1),
('b','NCO','bicelles_new_nc.tbl' ,.05),
('b','HNC','bicelles_new_hnc.tbl',.108)
]:
rdc = create_RDCPot("%s_%s"%(medium,expt),file,media[medium])

#1) scale prefactor relative to NH
# see python/rdcPotTools.py for exact calculation
# scale_toNH(rdc) - not needed for these datasets -
# but non-NH reported rmsd values will be wrong.

#3) Da rescaling factor (separate multiplicative factor)
# scale *= ( 9.9 / rdcs[name].oTensor.Da(0) )**2
rdc.setScale(scale)
rdc.setShowAllRestraints(1) #all restraints are printed during analysis
rdc.setThreshold(1.5) # in Hz
rdcs.append(rdc)
pass
potList.append(rdcs)
rampedParams.append( MultRamp(0.05,5.0, "rdcs.setScale( VALUE )") )
</pre>
The command to run python script is
<pre>xplor -py refine.py </pre>
== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/12565051?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=3 1.    Schwieters, C.D., Kuszewski, J.J., Tjandra, N. and Clore, G.M.  (2003) The Xplor-NIH NMR molecular structure determination package.  ''J. Magn. Res. 160'', 65-73.]

Residual Dipolar Couplings in Structure Refinement

2012-05-11T17:00:13Z

Lmorris: Undo revision 4093 by Lmorris (Talk)

== '''Introduction''' ==

Residual Dipolar Couplings (RDCs) originate from the anisotropic component of the dipolar interaction, which is dependent on the angle between an internuclear vector and the magnetic field. When a molecule sample orientations uniformly, as it does in normal solution NMR, RDCs average to zero and are not observable. However, if a molecule is dissolved in a dilute liquid crystalline medium it becomes partially aligned, and the dipolar couplings are not completely averaged to zero, leading to a small contribution to the splittings of NMR signals. The angular dependence of these contributions can provide valuable structural information. Protein structures can be validated using RDCs, and structures can be refined to improve quality.

Another application of RDCs is identification of the correct monomer orientations in homodimers. This application requires RDCs from two or more alignment media that give different orientations of principal alignment axes.

Below are protocols for the refinement of protein structures with RDCs using XPLOR-NIH [1].

 

== '''RDC Refinement Using XPLOR-NIH''' ==

=== Using XPLOR-NIH ===

X-PLOR can be downloaded from [http://nmr.cit.nih.gov/xplor-nih/ http://nmr.cit.nih.gov/xplor-nih/]. 

 

*Obtain a good estimate of the magnitude of Da and R from the alignment tensors. The alignment tensors can be obtained either from the extremes of distribution of RDCs (which forms a histogram) or from “REDCAT”. REDCAT gives values for Sxx, Syy and Szz. These values are relateive to the maxium RDC for that bond type. First, multiply these values from REDCAT by the associated maxium RDC value (for example 24350 for NH dipolar couplings). These absolute alignment tensors are related to Da and R as follows:
<pre>Da = Szz/2
R=Dr/Da where Dr=0.5*(Sxx-Syy)
</pre>
*Note that the sani constraint in XPLOR and or CNS requires three coefficients: DFS, Da and R. DFS is a fixed offset (for example a scalar coupling). For RDC data, DFS = 0.
*In XPLOR define the axis representing the alignment tensor coordinate system. The coordinate system is represented by four pseudo atoms: OO(origin), X ,Y and Z. The coordinate system has to be positioned far away to prevent any interaction with the protein. For more than one aligned media, define separate axes for each medium. These axes can either be defined at the end of the pdb file or as a separate .pdb file. Shown below is an example from the pdb:
<pre>ATOM 608 X ANI 500 33.000 30.000 30.000 1.00 0.00
ATOM 609 Y ANI 500 30.000 33.000 30.000 1.00 0.00
ATOM 610 Z ANI 500 30.000 30.000 33.000 1.00 0.00
ATOM 611 OO ANI 500 30.000 30.000 30.000 1.00 0.00
END
</pre>
You can also create a separate, standalone pdb file ('axis.pdb') with only one residue starting with ATOM 1. This separate pdb file can then be read in XPLOR or CNS as an additional coordinate file using the <tt>coor</tt> command.

 

*Create an RDC constraint file. An example is shown below where -8.1 is the coupling value and 0.6 and 0.5 are the errors associated with it:
<pre>assign ( resid 500 and name OO )
( resid 500 and name Z )
( resid 500 and name X )
( resid 500 and name Y )
( resid 1 and name HN )
( resid 1 and name N ) -8.1 0.6 0.5
</pre>
 

*The following perl script ('makeRdcAssign.pm') can be used to create RDC constraint file ('myprotRdc.tbl'). The input to the script is coupling values as one column text file (same as used in REDCAT to import rdcs). Usage for this script is <tt>makeRdcAssign.pm myprot.rdc myprotRdc.tbl startingResidue Err1 Err2</tt>. Same script can be used for CNS, the only change is to make a tbl file with only one error Err1.
<pre>#!/usr/bin/perl
if($ARGC < 3 ) {
print "Usage: makeRDCAssign.pm infile outfile startingResidue# <err1> <err2> \n";
}
$fileIn = $ARGV[0];
$fileOut = $ARGV[1];
$startRes = $ARGV[2];
$rdcE1 = 0.6;
$rdcE2 = 0.5;
$useE2 = 0;

if ($ARGV[3] ){
$rdcE1 = $ARGV[3];
}
if ($ARGV[4] ){
$rdcE2 = $ARGV[4];
$useE2 = 1;
}
unless( open(RDC_IN, $fileIn)) {
die("could not open input file $fileIn \n");
}
unless( open(RDC_OUT, ">$fileOut")) {
die("could not open outpu file $fileOut \n");
}

$thisRes = $startRes;
while ($thisLine = <RDC_IN> ) {
@parts = split(/\s+/,$thisLine ); # split by spaces
$thisRDC = $parts[0];
print("residue $thisRes, rdc $thisRDC \n");

# if the rdc is valid, write to the tbl file
if ($thisRDC < 999 ) {
print RDC_OUT "assign ( resid 500 and name OO ) \n";
print RDC_OUT " ( resid 500 and name Z ) \n";
print RDC_OUT " ( resid 500 and name X ) \n";
print RDC_OUT " ( resid 500 and name Y ) \n";
print RDC_OUT " ( resid $thisRes and name HN ) \n";
print RDC_OUT " ( resid $thisRes and name N ) ";
if ($useE2 > 0 ) {
print RDC_OUT " $thisRDC $rdcE1 $rdcE2 \n\n";
} else {
print RDC_OUT " $thisRDC $rdcE1 \n\n";
}
}
$thisRes = $thisRes + 1;
}
</pre>
 

*Do not include all the RDCs for refinement. Leave 15-20% of the RDCs for validation. 

*Set the Xplor-NIH input file for dipolar coupling refinement. The RDC restraint is known as sani (susceptibility anisotropy). Define the force constant, ksani for each media and for each type of couplings. 

*The refined structure can be validated with the RDCs that are not used in refinement. From the refined structure RDCs can be back calculated (using REDCAT) and plotted against the experimental RDCs. 

 

=== Details for performing RDC refinement in XPLOR/NIH starting from CNS ===

*Prepare the RDC constraint file as discussed above.
*Prepare noe and dihedral angle constraint files as for CNS ("aco.tbl", noe.tbl")
*The nomenclature and thus the topology of methyl groups in XPLOR is NOT the same as for CNS. You must rebuild each of your CNS pdb files into XPLOR format using the script <tt>generate1.inp</tt>. Edit the script and replace <tt>myprot</tt> with the appropriate prefix for your protein. generate.inp will also generate a structure file ("myprot.psf") for use with XPLOR. 
*The script "doi" will repeatedly run generate1.inp, iterating over all of your input structures.
*NOTE: generate1.inp does not like atoms OT1 and OT2 on the C terminal. Get rid of one and replace the other with O, make sure you preserve the exact column alignment in the pdb file.
*Refinment with RDCs sets anglular constraints relative to a virtual "axis" residue. You must also have a copy of the axis residue pdb file ("axis.pdb") in the local directory.
*The script refine_rdc_myprot.inp can be used to run the refinement including RDC constraints.
*You must edit the script to provide the correct names for your noe, dihedral and rdc input files xxx.tbl, you must also edit the coefficients for the NH sani constraints to put in your Da and Rhombicity as calculated above.
*The script "dos" will repeatedly run refine_rdc_myprot.inp, iterating over all of your input structures and creating refined structures.
*These refined structures may then be directly used in CNS if water bath refinement is desired.

 

=== Details for performing RDC refinement in XPLOR/NIH using Python Interface ===

XPLOR-NIH can be used by python interface. It does offer many conveniences over the old XPLOR script interface, for instance, no longer there is need to setup the .psf file for the alignment tensor and the value of Da and R don't have to be fixed. The details for the usage can be found on the tutorial attached. With the new version of XPLOR-NIH refine.py script can be found in the eginput directory. The script can be modified based on the number of media, values of Da and R. Below is the sani part of the script for the refrence. 
<pre># orientation Tensor - used with the dipolar coupling term
# one for each medium
# For each medium, specify a name, and initial values of Da, Rh.
#
from varTensorTools import create_VarTensor
media={}
# medium Da rhombicity
for (medium,Da,Rh) in [ ('t', -6.5, 0.62),
('b', -9.9, 0.23) ]:
oTensor = create_VarTensor(medium)
oTensor.setDa(Da)
oTensor.setRh(Rh)
media[medium] = oTensor
pass
# dipolar coupling restraints for protein amide NH.
# collect all RDCs in the rdcs PotList
# RDC scaling. Three possible contributions.
# 1) gamma_A * gamma_B / r_AB^3 prefactor. So that the same Da can be used
# for different expts. in the same medium. Sometimes the data is
# prescaled so that this is not needed. scale_toNH() is used for this.
# Note that if the expt. data has been prescaled, the values for rdc rmsd
# reported in the output will relative to the scaled values- not the expt.
# values.
# 2) expt. error scaling. Used here. A scale factor equal to 1/err^2
# (relative to that for NH) is used.
# 3) sometimes the reciprocal of the Da^2 is used if there is a large
# spread in Da values. Not used here.
#
from rdcPotTools import create_RDCPot, scale_toNH
rdcs = PotList('rdc')
for (medium,expt,file, scale) in \
[('t','NH' ,'tmv107_nh.tbl' ,1),
('t','NCO','tmv107_nc.tbl' ,.05),
('t','HNC','tmv107_hnc.tbl' ,.108),
('b','NH' ,'bicelles_new_nh.tbl' ,1),
('b','NCO','bicelles_new_nc.tbl' ,.05),
('b','HNC','bicelles_new_hnc.tbl',.108)
]:
rdc = create_RDCPot("%s_%s"%(medium,expt),file,media[medium])

#1) scale prefactor relative to NH
# see python/rdcPotTools.py for exact calculation
# scale_toNH(rdc) - not needed for these datasets -
# but non-NH reported rmsd values will be wrong.

#3) Da rescaling factor (separate multiplicative factor)
# scale *= ( 9.9 / rdcs[name].oTensor.Da(0) )**2
rdc.setScale(scale)
rdc.setShowAllRestraints(1) #all restraints are printed during analysis
rdc.setThreshold(1.5) # in Hz
rdcs.append(rdc)
pass
potList.append(rdcs)
rampedParams.append( MultRamp(0.05,5.0, "rdcs.setScale( VALUE )") )
</pre>
The command to run python script is
<pre>xplor -py refine.py </pre>
== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/12565051?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=3 1.    Schwieters, C.D., Kuszewski, J.J., Tjandra, N. and Clore, G.M.  (2003) The Xplor-NIH NMR molecular structure determination package.  ''J. Magn. Res. 160'', 65-73.]

Residual Dipolar Couplings in Structure Refinement

2012-05-11T16:58:31Z

Lmorris:

== '''Introduction''' == Residual Dipolar Couplings (RDCs) originate from partial anisotropic averaging of the dipolar interaction which is dependent on the angle between an internuclear vector and the magnetic field. When a molecule sample orientations uniformly, as it does in normal solution NMR, RDCs average to zero and are not observable. However, if a molecule is dissolved in a dilute liquid crystalline medium it becomes partially aligned, and the dipolar couplings are not completely averaged to zero, leading to a small contribution to the splittings of NMR signals. The angular dependence of these contributions can provide valuable structural information. Protein structures can be validated using RDCs, and structures can be refined to improve quality. Another application of RDCs is identification of the correct monomer orientation in homodimers. This application requires RDCs from two or more alignment media that give different orientations of principal alignment axes. Below are protocols for the refinement of protein structures with RDCs using XPLOR-NIH [1]. == '''RDC Refinement Using XPLOR-NIH''' == === Using XPLOR-NIH === X-PLOR can be downloaded from [http://nmr.cit.nih.gov/xplor-nih/ http://nmr.cit.nih.gov/xplor-nih/]. *Obtain a good estimate of the magnitude of Da and R from the alignment tensors. The alignment tensors can be obtained either from the extremes of distribution of RDCs (which forms a histogram) or from the program REDCAT. REDCAT gives values for Sxx, Syy and Szz. These values are relative to the maxium RDC (RDCmax) for that bond type. First, multiply these values from REDCAT by the associated maxium RDC value (for example 24350 for NH dipolar couplings). These alignment parameters are related to Da and R as follows:
<pre>Da = RDCmax * Szz/2
R = Dr/Da where Dr = 0.5*(Sxx-Syy) * RDCmax
</pre>
*Note that the sani constraint in XPLOR and or CNS requires three coefficients: DFS, Da and R. DFS is a fixed offset. For RDC data, DFS = 0. *In XPLOR define the axis representing the alignment tensor coordinate system. The coordinate system is represented by four pseudo atoms: OO(origin), X ,Y and Z. The coordinate system has to be positioned far away to prevent any interaction with the protein. For more than one aligned media, define separate axes for each medium. These axes can either be defined at the end of the pdb file or as a separate .pdb file. Shown below is an example from the pdb:
<pre>ATOM 608 X ANI 500 33.000 30.000 30.000 1.00 0.00
ATOM 609 Y ANI 500 30.000 33.000 30.000 1.00 0.00
ATOM 610 Z ANI 500 30.000 30.000 33.000 1.00 0.00
ATOM 611 OO ANI 500 30.000 30.000 30.000 1.00 0.00
END
</pre>
You can also create a separate, standalone pdb file ('axis.pdb') with only one residue starting with ATOM 1. This separate pdb file can then be read in XPLOR or CNS as an additional coordinate file using the <tt>coor</tt> command. *Create an RDC constraint file. An example is shown below where -8.1 is the coupling value with an associated error of plus and minus 0.5:
<pre>assign ( resid 500 and name OO )
( resid 500 and name Z )
( resid 500 and name X )
( resid 500 and name Y )
( resid 1 and name HN )
( resid 1 and name N ) -8.1 0.5 0.5

</pre>
*The following perl script ('makeRdcAssign.pm') can be used to create an RDC constraint file ('myprotRdc.tbl'). The input to the script is coupling values as one column text file (same as used in REDCAT to import rdcs). Usage for this script is: <tt>makeRdcAssign.pm myprot.rdc myprotRdc.tbl startingResidue Err1 Err2</tt>. The same script can be used for CNS, the only change is to make a tbl file with only one error Err1.
<pre>#!/usr/bin/perl
if($ARGC < 3 ) {
print "Usage: makeRDCAssign.pm infile outfile startingResidue# <err1> <err2> \n";
}
$fileIn = $ARGV[0];
$fileOut = $ARGV[1];
$startRes = $ARGV[2];
$rdcE1 = 0.6;
$rdcE2 = 0.5;
$useE2 = 0;

if ($ARGV[3] ){
$rdcE1 = $ARGV[3];
}
if ($ARGV[4] ){
$rdcE2 = $ARGV[4];
$useE2 = 1;
}
unless( open(RDC_IN, $fileIn)) {
die("could not open input file $fileIn \n");
}
unless( open(RDC_OUT, ">$fileOut")) {
die("could not open outpu file $fileOut \n");
}

$thisRes = $startRes;
while ($thisLine = <RDC_IN> ) {
@parts = split(/\s+/,$thisLine ); # split by spaces
$thisRDC = $parts[0];
print("residue $thisRes, rdc $thisRDC \n");

# if the rdc is valid, write to the tbl file
if ($thisRDC < 999 ) {
print RDC_OUT "assign ( resid 500 and name OO ) \n";
print RDC_OUT " ( resid 500 and name Z ) \n";
print RDC_OUT " ( resid 500 and name X ) \n";
print RDC_OUT " ( resid 500 and name Y ) \n";
print RDC_OUT " ( resid $thisRes and name HN ) \n";
print RDC_OUT " ( resid $thisRes and name N ) ";
if ($useE2 > 0 ) {
print RDC_OUT " $thisRDC $rdcE1 $rdcE2 \n\n";
} else {
print RDC_OUT " $thisRDC $rdcE1 \n\n";
}
}
$thisRes = $thisRes + 1;
}
</pre>
 *Do not include all the RDCs for refinement. Leave 15-20% of the RDCs for validation. *Set the Xplor-NIH input file for dipolar coupling refinement. The RDC restraint is known as sani (susceptibility anisotropy). Define the force constant, ksani for each media and for each type of couplings. *The refined structure can be validated with the RDCs that are not used in refinement. From the refined structure RDCs can be back calculated (using REDCAT) and plotted against the experimental RDCs. === Details for performing RDC refinement in XPLOR/NIH starting from CNS === *Prepare the RDC constraint file as discussed above. *Prepare noe and dihedral angle constraint files as for CNS ("aco.tbl", noe.tbl") *The nomenclature and thus the topology of methyl groups in XPLOR is NOT the same as for CNS. You must rebuild each of your CNS pdb files into XPLOR format using the script <tt>generate1.inp</tt>. Edit the script and replace <tt>myprot</tt> with the appropriate prefix for your protein. generate.inp will also generate a structure file ("myprot.psf") for use with XPLOR. *The script "doi" will repeatedly run generate1.inp, iterating over all of your input structures. *NOTE: generate1.inp does not like atoms OT1 and OT2 on the C terminal. Get rid of one and replace the other with O, make sure you preserve the exact column alignment in the pdb file. *Refinment with RDCs sets anglular constraints relative to a virtual "axis" residue. You must also have a copy of the axis residue pdb file ("axis.pdb") in the local directory. *The script refine_rdc_myprot.inp can be used to run the refinement including RDC constraints. *You must edit the script to provide the correct names for your noe, dihedral and rdc input files xxx.tbl, you must also edit the coefficients for the NH sani constraints to put in your Da and Rhombicity as calculated above. *The script "dos" will repeatedly run refine_rdc_myprot.inp, iterating over all of your input structures and creating refined structures. *These refined structures may then be directly used in CNS if water bath refinement is desired. === Details for performing RDC refinement in XPLOR/NIH using Python Interface === XPLOR-NIH can be used by python interface. It does offer many conveniences over the old XPLOR script interface, for instance, no longer there is need to setup the .psf file for the alignment tensor and the value of Da and R don't have to be fixed. The details for the usage can be found on the tutorial attached. With the new version of XPLOR-NIH refine.py script can be found in the eginput directory. The script can be modified based on the number of media, values of Da and R. Below is the sani part of the script for the refrence.
<pre># orientation Tensor - used with the dipolar coupling term
# one for each medium
# For each medium, specify a name, and initial values of Da, Rh.
#
from varTensorTools import create_VarTensor
media={}
# medium Da rhombicity
for (medium,Da,Rh) in [ ('t', -6.5, 0.62),
('b', -9.9, 0.23) ]:
oTensor = create_VarTensor(medium)
oTensor.setDa(Da)
oTensor.setRh(Rh)
media[medium] = oTensor
pass
# dipolar coupling restraints for protein amide NH.
# collect all RDCs in the rdcs PotList
# RDC scaling. Three possible contributions.
# 1) gamma_A * gamma_B / r_AB^3 prefactor. So that the same Da can be used
# for different expts. in the same medium. Sometimes the data is
# prescaled so that this is not needed. scale_toNH() is used for this.
# Note that if the expt. data has been prescaled, the values for rdc rmsd
# reported in the output will relative to the scaled values- not the expt.
# values.
# 2) expt. error scaling. Used here. A scale factor equal to 1/err^2
# (relative to that for NH) is used.
# 3) sometimes the reciprocal of the Da^2 is used if there is a large
# spread in Da values. Not used here.
#
from rdcPotTools import create_RDCPot, scale_toNH
rdcs = PotList('rdc')
for (medium,expt,file, scale) in \
[('t','NH' ,'tmv107_nh.tbl' ,1),
('t','NCO','tmv107_nc.tbl' ,.05),
('t','HNC','tmv107_hnc.tbl' ,.108),
('b','NH' ,'bicelles_new_nh.tbl' ,1),
('b','NCO','bicelles_new_nc.tbl' ,.05),
('b','HNC','bicelles_new_hnc.tbl',.108)
]:
rdc = create_RDCPot("%s_%s"%(medium,expt),file,media[medium])

#1) scale prefactor relative to NH
# see python/rdcPotTools.py for exact calculation
# scale_toNH(rdc) - not needed for these datasets -
# but non-NH reported rmsd values will be wrong.

#3) Da rescaling factor (separate multiplicative factor)
# scale *= ( 9.9 / rdcs[name].oTensor.Da(0) )**2
rdc.setScale(scale)
rdc.setShowAllRestraints(1) #all restraints are printed during analysis
rdc.setThreshold(1.5) # in Hz
rdcs.append(rdc)
pass
potList.append(rdcs)
rampedParams.append( MultRamp(0.05,5.0, "rdcs.setScale( VALUE )") )
</pre>
The command to run python script is
<pre>xplor -py refine.py </pre>
== '''References''' == [http://www.ncbi.nlm.nih.gov/pubmed/12565051?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=3 1.    Schwieters, C.D., Kuszewski, J.J., Tjandra, N. and Clore, G.M.  (2003) The Xplor-NIH NMR molecular structure determination package.  ''J. Magn. Res. 160'', 65-73.]

Alignment Media Preparation

2012-04-23T17:54:27Z

Lmorris:

=== Brief Description ===

NH RDC are easily acquired and for the purpose of protein structure validation and refinement and the data can be collected on samples labeled only with 15N. Obtaining RDC in two different alignment media greatly improve the quality of refinement and can aid in dimer structure determination; 500ul of a 0.5-0.6mM sample is usually sufficient for this purpose.

=== Data Acquisition to RDC Calculation ===

Detail description on data acquisition

=== Alignment Media Preparation ===

==== Isotropic Sample Measurement ====

The first sample to be observed is an isotropic sample. Dilute one half of the sample by a factor of two as this will give a reference at the concentration of most aligned samples. This can be important in cases of weak dimers. Measure the pH as this will be useful in selecting media later.

==== PEG Biclle ====

Alignment of the protein sample in PEG(C12E5/hexanol). This is used as a first alignement media because it produced primarily steric alignment (useful in dimer geometry predictions), the success rate is high, and it can be doped with other charged detergents to give a second alignment media .

Chemical used for this preparation:

:'''Sigma Aldrich 76437''', Pentaethylene glycol monododecyl ether (C12E5 PEG)
:'''Sigma Aldrich H13303''', Hexanol
:'''Sigma Aldrich 855820''', Cetyltrimethylammonium bromide (CTAB)
:'''Sigma Aldrich O4003''', Sodium octyl sulfate (SOS)
:'''Sigma Aldrich 436143''', Sodium dodecyl sulfate (SDS)

The preparation procedure is as follow:

*Mix 50ul of C12E5 (pentaethylene glycol monododecyl ether) with 200ul of buffer and 50ul of D2O by vortexing (REF <ref> Ruckert M and Otting G (2000), JACS, 122, 7793-7797</ref>).
*Add approx 16ul of hexanol, in aliquots of 2ul with vortexing after each addition. The solution will go from clear to milky, then to translucent and viscous with lots of bubbles. Continue to add hexanol until the solution goes clear again. If it becomes milky/turbid again, you have gone past the nematic phase.
*Sample Content:

::155 uL of Protein
::55 uL of 16% PEG stock solution
::20 uL of D2O
:::Final PEG concentration is 4.2%.

*Record the 2H splitting by running the s2pul expt with tn=lk (for Varian instrument). The range of the splitting should be around ~+/-20Hz.

*PEG can be doped with either cetyltrimethyl ammonium bromide (CTAB) for positively charged proteins or sodium octyl sulphate (SOS) for negatively charged proteins. Charging the medium to be like the protein prevents association and gives higher resolution spectra. A suitable ratio of PEG:CTAB/SOS is ~30:1.

==== Pf1 Phage ====

Preparation of a Pf1 phage alignment sample is fairly straightforward (REF<ref>Hansen MR, Mueller L, Pardi A (1998), Nat Struct Biol, 5, 1065-1074</ref>). The protein sample is diluted by the alignment medium.

Chemical used for this preparation:

:'''ASLA Biotech P-50-P''', Pf1 phage 50 mg/mL

The preparation procedure is as follows:

*Start with a protein stock 1-2 mM and a pf1 phage stock of 50 mg/mL. Prepare a sample of 12.5 mg/mL of phage.

*Sample Content:

::155 uL of Protein
::55 uL of Pf1 phage
::20 uL of D2O
:::Final phage concentration is 12.5 mg/mL

*Record the 2H splitting by running the s2pul expt with tn=lk (for Varian instrument). The range of the splitting should be around ~+/-8~10 Hz.

==== Polyacrylamide Gel (Compressed and Stretched) ====

Chemicals used in preparing this alignment medium can are:

:'''Bio-Rad 161-0144''', 40%Acrylamide/Bis solution 19:1
:'''Bio-Rad 161-0733''', 10X TBE
:'''Bio-Rad 161-0700''', APS
:'''Bio-Rad 161-0800''', TEMED
:'''Sigma Aldrich M7279-25G''', N,N'-methylenebisacrylamide (BIS)
:'''Sigma Aldrich 448281''', (3-Acrylamidopropyl)trimethylammonium chloride solution
:'''Sigma Aldrich 282731''', 2-Acrylamido-2-methyl-1-propanesulfonic acid

===== Compressed Gels - Positive, Negative and Neutral =====

*Mix stock solutions of 40% acrylamide and N,N'-methylenebisacrylamide in a 19:1 ratio with 40% charged acrylate derivatives containing N,N'-methylenebisacrylamide as desired in a 19:1 ratio.

*Dilute the mixtures 10x with TBE buffer (0.9 M TRIS, 0.9 M borate, 0.02 M EDTA, pH 8.2) to a final 7% concentration.

*Polymerization is initiated by the addition of 0.15% ammoniumperoxide sulfate and 1% tetramethylethylenediamine (TEMED).

*To introduce negative charges, use acrylic acid or 2-acrylamido-2-methyl-1-propanesulfonic acid. Positive charge can be introduced by addition of (3-acrylamidopropyl)-trimethylammonium chloride (APTMAC) or N-(2-acryloamidoethyl) triethylammonium iodide. For neutral gels omit the charged species; for zwitterionic gels use equivalent amounts of acrylic acid and APTMAC. For 50% charged gels use 50% content of the charged specie and 50% acrylamide.

*Add 130ul of the mixture to plastic tubes with a 3.2 mm inner diameter and keep them overnight allowing polymerization to occur.

*Wash the polymerized gels extensively in deionized water (three cycles over a period of 2 days). The gels will increase in size due to electro osmotic swelling.

*Dry the gels over a 2 day period at room temperature on a teflon pan.

=== References ===

<references /> Updated by Hsiau-Wei Lee, 2011

Alignment Media Preparation

2012-04-23T17:53:15Z

Lmorris:

=== Brief Description ===

NH RDC are easily acquired and for the purpose of protein structure validation and refinement and the data can be collected on samples labeled only with 15N. Obtaining RDC in two different alignment media greatly improve the quality of refinement and can aid in dimer structure determination; 500ul of a 0.5-0.6mM sample is usually sufficient for this purpose.

=== Data Acquisition to RDC Calculation ===

Detail description on data acquisition

=== Alignment Media Preparation ===

==== Isotropic Sample Measurement ====

The first sample to be observed is an isotropic sample. Dilute one half of the sample by a factor of two as this will give a reference at the concentration of most aligned samples. This can be important in cases of weak dimers. Measure the pH as this will be useful in selecting media later.

==== PEG Biclle ====

Alignment of the protein sample in PEG(C12E5/hexanol). This is used as a first alignement media because it produced primarily steric alignment (useful in dimer geometry predictions), the success rate is high, and it can be doped with other charged detergents to give a second alignment media .

Chemical used for this preparation:

:'''Sigma Aldrich 76437''', Pentaethylene glycol monododecyl ether (C12E5 PEG)
:'''Sigma Aldrich H13303''', Hexanol
:'''Sigma Aldrich 855820''', Cetyltrimethylammonium bromide (CTAB)
:'''Sigma Aldrich O4003''', Sodium octyl sulfate (SOS)
:'''Sigma Aldrich 436143''', Sodium dodecyl sulfate (SDS)

The preparation procedure is as follow:

*Mix 50ul of C12E5 (pentaethylene glycol monododecyl ether) with 200ul of buffer and 50ul of D2O by vortexing (REF <ref> Ruckert M and Otting G (2000), JACS, 122, 7793-7797</ref>).
*Add approx 16ul of hexanol, in aliquots of 2ul with vortexing after each addition. The solution will go from clear to milky, then to translucent and viscous with lots of bubbles. Continue to add hexanol until the solution goes clear again. If it becomes milky/turbid again, you have gone past the nematic phase.
*Sample Content:

::155 uL of Protein
::55 uL of 16% PEG stock solution
::20 uL of D2O
:::Final PEG concentration is 4.2%.

*Record the 2H splitting by running the s2pul expt with tn=lk (for Varian instrument). The range of the splitting should be around ~+/-20Hz.

*PEG can be doped with either cetyltrimethyl ammonium bromide (CTAB) for positively charged proteins or sodium octyl sulphate (SOS) for negatively charged proteins. Charging the medium to be like the protein prevents association and gives higher resolution spectra. A suitable ratio of PEG:CTAB/SOS is ~30:1.

==== Pf1 Phage ====

Preparation of a Pf1 phage alignment sample is fairly straightforward (REF<ref>Hansen MR, Mueller L, Pardi A (1998), Nat Struct Biol, 5, 1065-1074</ref>). The protein sample is diluted by the alignment medium.

Chemical used for this preparation:

:'''ASLA Biotech P-50-P''', Pf1 phage 50 mg/mL

The preparation procedure is as follows:

*Start with a protein stock 1-2 mM and a pf1 phage stock of 50 mg/mL. Prepare a sample of 12.5 mg/mL of phage.

*Sample Content:

::155 uL of Protein
::55 uL of Pf1 phage
::20 uL of D2O
:::Final phage concentration is 12.5 mg/mL

*Record the 2H splitting by running the s2pul expt with tn=lk (for Varian instrument). The range of the splitting should be around ~+/-8~10 Hz.

==== Polyacrylamide Gel (Compress and Stretch) ====

Chemicals used in preparing this alignment medium can are:

:'''Bio-Rad 161-0144''', 40%Acrylamide/Bis solution 19:1
:'''Bio-Rad 161-0733''', 10X TBE
:'''Bio-Rad 161-0700''', APS
:'''Bio-Rad 161-0800''', TEMED
:'''Sigma Aldrich M7279-25G''', N,N'-methylenebisacrylamide (BIS)
:'''Sigma Aldrich 448281''', (3-Acrylamidopropyl)trimethylammonium chloride solution
:'''Sigma Aldrich 282731''', 2-Acrylamido-2-methyl-1-propanesulfonic acid

===== Compressed Gels can be made Positive, Negative and Neutral =====

*Mix stock solutions of 40% acrylamide and N,N'-methylenebisacrylamide in a 19:1 ratio with 40% charged acrylate derivatives containing N,N'-methylenebisacrylamide as desired in a 19:1 ratio.

*Dilute the mixtures 10x with TBE buffer (0.9 M TRIS, 0.9 M borate, 0.02 M EDTA, pH 8.2) to a final 7% concentration.

*Polymerization is initiated by the addition of 0.15% ammoniumperoxide sulfate and 1% tetramethylethylenediamine (TEMED).

*To introduce negative charges, use acrylic acid or 2-acrylamido-2-methyl-1-propanesulfonic acid. Positive charge can be introduced by addition of (3-acrylamidopropyl)-trimethylammonium chloride (APTMAC) or N-(2-acryloamidoethyl) triethylammonium iodide. For neutral gels omit the charged species; for zwitterionic gels use equivalent amounts of acrylic acid and APTMAC. For 50% charged gels use 50% content of the charged specie and 50% acrylamide.

*Add 130ul of the mixture to plastic tubes with a 3.2 mm inner diameter and keep them overnight allowing polymerization to occur.

*Wash the polymerized gels extensively in deionized water (three cycles over a period of 2 days). The gels will increase in size due to electro osmotic swelling.

*Dry the gels over a 2 day period at room temperature on a teflon pan.

=== References ===

<references /> Updated by Hsiau-Wei Lee, 2011

Residual Dipolar Couplings in Structure Refinement

2012-02-21T17:21:27Z

Lmorris: Undo revision 4000 by Lmorris (Talk)

Residual Dipolar Couplings in Structure Refinement

2012-02-21T17:20:49Z

Lmorris:

== '''Introduction''' ==

Residual Dipolar Couplings (RDCs) originate from the anisotropic component of the dipolar interaction, which is dependent on the angle between an internuclear vector and the magnetic field. When a molecule sample orientations uniformly, as it does in normal solution NMR, RDCs average to zero and are not observable. However, if a molecule is dissolved in a dilute liquid crystalline medium it becomes partially aligned, and the dipolar couplings are not completely averaged to zero, leading to a small contribution to the splittings of NMR signals. The angular dependence of these contributions can provide valuable structural information. Protein structures can be validated using RDCs, and structures can be refined to improve quality.

Another application of RDCs is identification of the correct monomer orientations in homodimers. This application requires RDCs from two or more alignment media that give different orientations of principal alignment axes.

Below are protocols for the refinement of protein structures with RDCs using XPLOR-NIH [1]. 2 3 4

 

== '''RDC Refinement Using XPLOR-NIH''' ==

=== Using XPLOR-NIH ===

X-PLOR can be downloaded from [http://nmr.cit.nih.gov/xplor-nih/ http://nmr.cit.nih.gov/xplor-nih/]. 

 

*Obtain a good estimate of the magnitude of Da and R from the alignment tensors. The alignment tensors can be obtained either from the extremes of distribution of RDCs (which forms a histogram) or from “REDCAT”. REDCAT gives values for Sxx, Syy and Szz. These values are relateive to the maxium RDC for that bond type. First, multiply these values from REDCAT by the associated maxium RDC value (for example 24350 for NH dipolar couplings). These absolute alignment tensors are related to Da and R as follows:
<pre>Da = Szz/2
R=Dr/Da where Dr=0.5*(Sxx-Syy)
</pre>
*Note that the sani constraint in XPLOR and or CNS requires three coefficients: DFS, Da and R. DFS is a fixed offset (for example a scalar coupling). For RDC data, DFS = 0.
*In XPLOR define the axis representing the alignment tensor coordinate system. The coordinate system is represented by four pseudo atoms: OO(origin), X ,Y and Z. The coordinate system has to be positioned far away to prevent any interaction with the protein. For more than one aligned media, define separate axes for each medium. These axes can either be defined at the end of the pdb file or as a separate .pdb file. Shown below is an example from the pdb:
<pre>ATOM 608 X ANI 500 33.000 30.000 30.000 1.00 0.00
ATOM 609 Y ANI 500 30.000 33.000 30.000 1.00 0.00
ATOM 610 Z ANI 500 30.000 30.000 33.000 1.00 0.00
ATOM 611 OO ANI 500 30.000 30.000 30.000 1.00 0.00
END
</pre>
You can also create a separate, standalone pdb file ('axis.pdb') with only one residue starting with ATOM 1. This separate pdb file can then be read in XPLOR or CNS as an additional coordinate file using the <tt>coor</tt> command.

 

*Create an RDC constraint file. An example is shown below where -8.1 is the coupling value and 0.6 and 0.5 are the errors associated with it:
<pre>assign ( resid 500 and name OO )
( resid 500 and name Z )
( resid 500 and name X )
( resid 500 and name Y )
( resid 1 and name HN )
( resid 1 and name N ) -8.1 0.6 0.5
</pre>
 

*The following perl script ('makeRdcAssign.pm') can be used to create RDC constraint file ('myprotRdc.tbl'). The input to the script is coupling values as one column text file (same as used in REDCAT to import rdcs). Usage for this script is <tt>makeRdcAssign.pm myprot.rdc myprotRdc.tbl startingResidue Err1 Err2</tt>. Same script can be used for CNS, the only change is to make a tbl file with only one error Err1.
<pre>#!/usr/bin/perl
if($ARGC < 3 ) {
print "Usage: makeRDCAssign.pm infile outfile startingResidue# <err1> <err2> \n";
}
$fileIn = $ARGV[0];
$fileOut = $ARGV[1];
$startRes = $ARGV[2];
$rdcE1 = 0.6;
$rdcE2 = 0.5;
$useE2 = 0;

if ($ARGV[3] ){
$rdcE1 = $ARGV[3];
}
if ($ARGV[4] ){
$rdcE2 = $ARGV[4];
$useE2 = 1;
}
unless( open(RDC_IN, $fileIn)) {
die("could not open input file $fileIn \n");
}
unless( open(RDC_OUT, ">$fileOut")) {
die("could not open outpu file $fileOut \n");
}

$thisRes = $startRes;
while ($thisLine = <RDC_IN> ) {
@parts = split(/\s+/,$thisLine ); # split by spaces
$thisRDC = $parts[0];
print("residue $thisRes, rdc $thisRDC \n");

# if the rdc is valid, write to the tbl file
if ($thisRDC < 999 ) {
print RDC_OUT "assign ( resid 500 and name OO ) \n";
print RDC_OUT " ( resid 500 and name Z ) \n";
print RDC_OUT " ( resid 500 and name X ) \n";
print RDC_OUT " ( resid 500 and name Y ) \n";
print RDC_OUT " ( resid $thisRes and name HN ) \n";
print RDC_OUT " ( resid $thisRes and name N ) ";
if ($useE2 > 0 ) {
print RDC_OUT " $thisRDC $rdcE1 $rdcE2 \n\n";
} else {
print RDC_OUT " $thisRDC $rdcE1 \n\n";
}
}
$thisRes = $thisRes + 1;
}
</pre>
 

*Do not include all the RDCs for refinement. Leave 15-20% of the RDCs for validation. 

*Set the Xplor-NIH input file for dipolar coupling refinement. The RDC restraint is known as sani (susceptibility anisotropy). Define the force constant, ksani for each media and for each type of couplings. 

*The refined structure can be validated with the RDCs that are not used in refinement. From the refined structure RDCs can be back calculated (using REDCAT) and plotted against the experimental RDCs. 

 

=== Details for performing RDC refinement in XPLOR/NIH starting from CNS ===

*Prepare the RDC constraint file as discussed above.
*Prepare noe and dihedral angle constraint files as for CNS ("aco.tbl", noe.tbl")
*The nomenclature and thus the topology of methyl groups in XPLOR is NOT the same as for CNS. You must rebuild each of your CNS pdb files into XPLOR format using the script <tt>generate1.inp</tt>. Edit the script and replace <tt>myprot</tt> with the appropriate prefix for your protein. generate.inp will also generate a structure file ("myprot.psf") for use with XPLOR. 
*The script "doi" will repeatedly run generate1.inp, iterating over all of your input structures.
*NOTE: generate1.inp does not like atoms OT1 and OT2 on the C terminal. Get rid of one and replace the other with O, make sure you preserve the exact column alignment in the pdb file.
*Refinment with RDCs sets anglular constraints relative to a virtual "axis" residue. You must also have a copy of the axis residue pdb file ("axis.pdb") in the local directory.
*The script refine_rdc_myprot.inp can be used to run the refinement including RDC constraints.
*You must edit the script to provide the correct names for your noe, dihedral and rdc input files xxx.tbl, you must also edit the coefficients for the NH sani constraints to put in your Da and Rhombicity as calculated above.
*The script "dos" will repeatedly run refine_rdc_myprot.inp, iterating over all of your input structures and creating refined structures.
*These refined structures may then be directly used in CNS if water bath refinement is desired.

 

=== Details for performing RDC refinement in XPLOR/NIH using Python Interface ===

XPLOR-NIH can be used by python interface. It does offer many conveniences over the old XPLOR script interface, for instance, no longer there is need to setup the .psf file for the alignment tensor and the value of Da and R don't have to be fixed. The details for the usage can be found on the tutorial attached. With the new version of XPLOR-NIH refine.py script can be found in the eginput directory. The script can be modified based on the number of media, values of Da and R. Below is the sani part of the script for the refrence. 
<pre># orientation Tensor - used with the dipolar coupling term
# one for each medium
# For each medium, specify a name, and initial values of Da, Rh.
#
from varTensorTools import create_VarTensor
media={}
# medium Da rhombicity
for (medium,Da,Rh) in [ ('t', -6.5, 0.62),
('b', -9.9, 0.23) ]:
oTensor = create_VarTensor(medium)
oTensor.setDa(Da)
oTensor.setRh(Rh)
media[medium] = oTensor
pass
# dipolar coupling restraints for protein amide NH.
# collect all RDCs in the rdcs PotList
# RDC scaling. Three possible contributions.
# 1) gamma_A * gamma_B / r_AB^3 prefactor. So that the same Da can be used
# for different expts. in the same medium. Sometimes the data is
# prescaled so that this is not needed. scale_toNH() is used for this.
# Note that if the expt. data has been prescaled, the values for rdc rmsd
# reported in the output will relative to the scaled values- not the expt.
# values.
# 2) expt. error scaling. Used here. A scale factor equal to 1/err^2
# (relative to that for NH) is used.
# 3) sometimes the reciprocal of the Da^2 is used if there is a large
# spread in Da values. Not used here.
#
from rdcPotTools import create_RDCPot, scale_toNH
rdcs = PotList('rdc')
for (medium,expt,file, scale) in \
[('t','NH' ,'tmv107_nh.tbl' ,1),
('t','NCO','tmv107_nc.tbl' ,.05),
('t','HNC','tmv107_hnc.tbl' ,.108),
('b','NH' ,'bicelles_new_nh.tbl' ,1),
('b','NCO','bicelles_new_nc.tbl' ,.05),
('b','HNC','bicelles_new_hnc.tbl',.108)
]:
rdc = create_RDCPot("%s_%s"%(medium,expt),file,media[medium])

#1) scale prefactor relative to NH
# see python/rdcPotTools.py for exact calculation
# scale_toNH(rdc) - not needed for these datasets -
# but non-NH reported rmsd values will be wrong.

#3) Da rescaling factor (separate multiplicative factor)
# scale *= ( 9.9 / rdcs[name].oTensor.Da(0) )**2
rdc.setScale(scale)
rdc.setShowAllRestraints(1) #all restraints are printed during analysis
rdc.setThreshold(1.5) # in Hz
rdcs.append(rdc)
pass
potList.append(rdcs)
rampedParams.append( MultRamp(0.05,5.0, "rdcs.setScale( VALUE )") )
</pre>
The command to run python script is
<pre>xplor -py refine.py </pre>
== '''References''' ==

[http://www.ncbi.nlm.nih.gov/pubmed/12565051?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=3 1.    Schwieters, C.D., Kuszewski, J.J., Tjandra, N. and Clore, G.M.  (2003) The Xplor-NIH NMR molecular structure determination package.  ''J. Magn. Res. 160'', 65-73.]