Homodimer Structure Calculation with Symmetry Constraints

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 and analysis of an automated structure determination run for symmertic homodimers starting from NOE peaklists in the framework of CYANA-3.0.

 Results from CYANA 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 Homodimer Structure Calculation Setup

The structure calculation with automated noesy assignments and intermolecular restraints follows the canonical CYANA recipe.  Simple annealing calculations 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), a CALC.cya script and an init.cya script.  

An helpful script called DIMERIZE, to be run from within CYANA, can be used to symmetrize various files such as the proton (.prot), angle and NOE constraint files:

!/bin/sh

# Chemical shift list
cp RpR324.prot RpR324d.prot
awk 'NF>=5 { printf("%6d%8.3f%8.3f %-5s%4d\n",$1+1000,$2,$3,$4,$5+200) }' $* RpR324.prot >> RpR324d.prot

# Additional distance constraints
echo "
read upl D5.upl
read lol D5.lol
molecules symmetrize
write upl D5dimer.upl
write lol D5dimer.lol
quit" | cyana32

# angle constraints
echo "
read dihe.aco
shift +200
writeaco t.aco
quit" | ancoma
cat dihe.aco t.aco > dihed.aco
rm -f t.aco

The sequence file

The example sequence file, shown below in abbreviated form, now includes the doubled sequence separated by a 48-element (LL2) linker:

MET      1
THR      2
SER      3
THR      4
PHE      5
ASP      6
.
.
.
HIS    100
HIS    101

PL     102
LL2    103
LL2    104
.
.
LL2    148
LL2    149
LP     150

MET    201
THR    202
SER    203
THR    204
PHE    205
ASP    206
.
.
.
HIS    300
HIS    301

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,xfilt.peaks  # names of NOESY peak lists
prot        := RpR324d.prot               # names of chemical shift lists
restraints  := ssa.cya,hbonds.upl,hbonds.lol,manual.upl,dihed.aco  # 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

ssa
noeassign peaks=$peaks prot=$prot autoaco

Defining inter-chain only peaks

Notice the peak contain a list of intermolecular NOEs called xfilt.peaks.  It is possible to instruct the program to make only interchain NOE assignments for the peaklist by using the 9 U xeasy color-code as shown below, the remaining peaklists can be treated as usual as a mixture of inter and intra chain NOEs.

# Number of dimensions 3
#FORMAT xeasy3D
#INAME 1 h
#INAME 2 C
#INAME 3 H
#CYANAFORMAT hCH
      1   8.072  35.125   4.110 9 U   1.000E+06  0.000E+00 e 0     0     0     0
      2   2.584  33.247   4.446 9 U   3.610E+05  0.000E+00 e 0     0     0     0
      3   6.801  42.287   3.199 9 U   4.280E+05  0.000E+00 e 0     0     0     0
      4   6.799  42.276   2.940 9 U   3.440E+05  0.000E+00 e 0     0     0     0 

The Init.cya file

The new intstructions for symmetric dimer treatment are included in the init.cya file, in the example below molecule indentity and molecule symmetry are parametrized and adjustable (probably the parameters have been optimized so results tweaking the weight_ide and weight_sym should be carefully).

name:=RpR324
rmsdrange:=10-80
cyanalib
nproc:=8
read seq $name.seq
molecules define 1..101 201..301
molecule identity
weight_ide=0.03
molecule symdist "CA 1..101" "CA 201..301"
weight_sym=0.0025

The weight of the symmetry and indentity constraints does not appear to be parametrized in the target function as a penalty.  Further explanation will hopefully become available with the official release.

The use of MPI routine is highly recommended due to the length of the calculation.

Output analysis

The output analysis is carried out in the usual manner as for CYANA -2.1.