Structure Calculation With RDC's Using CYANA
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 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):
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
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 weight factors:
# 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
Multiple media (e.g. orientations) should be listed as follows:
# Orientation Magnitude Rhombicity ORI residue number
1 5.39535 0.63125 360
2 7.55656 0.58200 370
and the sequence should be modified to include further links and ORI:
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
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.
## 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 = $rhombicity(i)." end do rdc fittensor method=simplex # (can take several minutes) #rdc fittensor method=gridsearch # systematic search (very slow) # alternatively, determine tensor from given structure by SVD read rdc phage_all_mono.rdc read pdb final.pdb overview
The dummy values in the rdc list are read initially and they can be updated after running the FindTensor.cya routine. If no models are present (e.g. final.pdb) the program will terminate with a warning.
The CALC.cya file
The working directory that contains all the files necessary to start the calcualtion is ready and the CALC.cya modified for the presence of RDC constraints is used:
peaks := ali5.peaks,aro5.peaks,n3.peaks # names of NOESY peak lists prot := RpR324.prot # names of chemical shift lists restraints := ssa.cya,phage_all_mono.rdc # additional (non-NOE) constraints tolerance := 0.04,0.025,0.3 # chemical shift tolerances: HX2-HX1-X1 calibration := # NOE calibration parameters structures := 100,20 # number of initial, final structures steps := 10000 # number of torsion angle dynamics steps rmsdrange := 10..80 # residue range for RMSD calculation randomseed := 56231 # random number generator seed weight_rdc = 0.02 # weight for RDC restraints cut_rdc = 0.2 # cutoff for RDC violation output ssa noeassign peaks=$peaks prot=$prot autoaco
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:
name:=RpR324 rmsdrange:=10-80 cyanalib nproc:=8 read seq $name.seq rdcdistances
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.
# 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."
Notes on run execution
The command line execution form single machine or cluster using MPI implememntation (highly recommended) is carried on as usual:
/cyana-3.0/cyana CALC > & log &
the MPI is launched using the script called, for example, submit_cyana:
#!/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
with the command:
qsub -q @master3 submit_cyana
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):
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)