MMTSB Tool Set - REX/GB Refinement of NMR Structures

Objective and Overview

This tutorial illustrates how to use the replica exchange (REX) molecular dynamics (MD) simulation together with the GBSW implicit solvent to refine NMR structures. This is a quite involved process that requires the following steps:

  • Convert the original NMR data file (*.mr) into CHARMM restrain files.
  • Prepare and submit a PBS job to carry out the REX refinement simulation in GBSW.
  • Analysis of the refinement resutls: extract and cluster the structure ensemble at the lowest temperature. Calculate the average structures of each cluster and do NMR restraint violation analysis.

The simulation will make extensive usage of various CHARMM analysis facilities and the MMTSB Tool Set. Several stand alone scripts are also used to convert data formats and assis analysis. Such combination of "standard" tools and custom scripts is typical in actual research. This particular example is actually based on an actual project that is now published (Chen et al., JBNMR, 2004, see PubMed). All required files including this page can be downloaded as a TAR/GZ file.

1. Iniitial Structures and NOE restraints

We will use the Zinc-dependent Redox Switch Domain of the Chaperone Hsp33. This protein domain has a novel fold and the NMR structure was solved by Jane Dyson's group at Scripps and is now available from the the Protein Data Bank with the PDB code 1XJH A minimized average structure of the NMR ensemble (hsp.ref.pdb) is provided.

During the early stages of structure determination, only a limit set of NOE distance restraints were assigned (see data/mrdata/ There were only 30 long-range NOEs, six of which define zinc coordination and the rest of which are between residues in the beta-hairpin. There is virturally no information about the tertiary fold and this is why the CYANA and CNS calculations failed to identify the tertiary fold (e.g., see above). A set of 16 representative structures computed by CNS are included directory "inipdb/". One can plot the residue NOE contacts using gnuplot,

     set grid
     plot 'data/mrdata/' u 4:11 w p
The plot will look like:

Residue-residue contact map of all assigned NOEs

The NOE data needs to be translated to CHARMM format. This was done using a simple perl script To run the script, type:

     cd data/mrdata
     ./scripts/ manual

The NOEs will be grouped into intra-residue (i=j), sequential (|i-j|=1), medium-range (|i-j|<=4) and long-range (|i-j|)>4) ones and written to separate files (manual.noe.*). The separation of different types of NOEs facilitate later restraint violation analysis.

2. REX/GB Refinement Simulation

Once the initial structures and the NOE distance restraint files ae ready. We need to do a few more twists in order to run the REX/GB refinement simulation.

First, we need to write "custom setup" file to tell how to set up the NMR restraints before running the MD simulations. This information is given in file "data/noe.str". This file will be provided to "" (see below).

     set mrbase = data/mrdata/manual.noe
     set fmax = 0.1
     set kmax = 1.0
     set kmin = 1.0
     set rswi = 3.0

     rename atom CD1  sele resn ILE .and. type CD  end
     bomlev -1
     stream @mrbase.intra
     stream @mrbase.seq
     stream @mrbase.long
     bomlev 0
     rename atom CD  sele resn ILE .and. type CD1  end

       SCALE 10
     mini sd   nstep 100 step 0.005 nprint 50
     mini abnr nstep 100 step 0.005 nprint 50
Note that the above script includes a short minimization to relax the CNS-generated initial PDB structures before calls for dynamics.

Second, this particular protein contains zinc ions and requires a special patch to the topology and paramter files (if your proteins do not have bound ions, this step will not be necessary). The modified topology and parameter files (with two new residue types, CYSS and CYSZ, as defined in CNS) are provided in directory "toppar/". In order for the MMTSB Tool Set to locate them, you either need to copy these two files to directory $CHARMMDATA, or, redefine $CHARMMDATA in your shell configuration file (e.g., .tcshrc). The first option is recommended to avoid messing up other REX tutorials.

Now we are ready to prepare a PBS script for running the REX/GB refinement simulation. In the script rex.pbs, we request 1000 cycles of REX simulation using 16 replicas. Each cycle contains 0.5 ps restrained MD and the job should take about 12 hours on 2.40GHz Xeon processors.

   #!/bin/csh -f
   #PBS -l nodes=8:ppn=2
   #PBS -l walltime=48:00:00
   #PBS -l cput=24:00:00
   #PBS -j eo
   #PBS -m ea


   cat $PBS_NODEFILE | sort -u | \
    awk '{ print $1, " 2 ",dir }' dir=$PBSTMPDIR >! host.$$ -mp -hosts  host.$$  -n 1000 -dir rexgb \
    -log rexserver.log -charmmlog rex.log \
    -mdpar param=22x,xtop=top22_zinc.inp,xpar=par22_zinc.inp \
    -mdpar dynsteps=250,dyntstep=0.002,cuton=16,cutoff=16,cutnb=20 \
    -mdpar gb=gbsw,gbswsgamma=0.01,gbswdgp=1.5,scalerad=nina,cmap=1 \
    -par archive,natpdb=hsp.ref.pdb \
    -custom setup data/noe.str -temp 16:300:600 inipdb/*.pdb


A few things need to be note in the above script.

1). There are muliple initial structures. will automatically assign each initial structure to a replica. In principle, one can also refine a single structure. However, in case of NMR structure calcuation, it is better to use a diverse ensemble of structures generated by CNS or DYANA, such that there is a higher chance of sampling the true native fold.

2). The NOE restraints are imposed during the REX/GB simulation through -custom setup noe.str option. will read the script noe.str before the dynamics runs for all replicas. The purpose here is to combine experimental data with modern GB force field to achieve better structural convergence than either GB or NMR alone.

3). Non-standard topology and parameter files are specified through -xtop, -xpar options

Finally(!), we are ready to submit the job (Note, remember to submit from directory "REX_NMRRefinement_Tutorial/files/"):

      qsub scripts/rex.pbs

Also note that the TAR/GZ file contains a dirctory called "rexgb_example". This is REX data directory produced by a previous simulation. All the archive, restart and backup files were deleted to save space. But the final snapshots and REX data structues are retained. It also contains a directory "ens0/", which is the ensemble of the last 200 snapshots sampled at the lowest temperature. "ens0/" also contains results from clustering and CHARMM analysis (see below).

3. Extracting, Clusterinng and Energy Minimization

During the course of the simulation, one can examine the convergence of energy using, -bycond 0 -dir rexgb
It is also a good idea to make sure that exchange acceptance ratio is sufficiently high, which is given in file rexgb/rexserver.ninx. When the job is finished, one can extract the structures sampled at the lowest temperature and cluster them to obtain refined models. Results of a previous run are included here (see data/rexgb_example). The energy converges after 600-700 REX steps, shown below:

1).Run following command under rexgb directory to extract the last 200 structures at the lowest ensemble,
    cd rexgb
    ../scripts/ -inx 801:1000
alternatively, one can also extract the ensemble using starndard MMSTB tools, -inx 801:1000 -bycond 0 \
                   -apply " -dir ens0 rex - "
2). Go to the directory ens0 that was just created, and cluster the structures using with default options,
    cd ens0 -l 7:62 rex
The results of clustering can be checked by, rex
3). Compute the average structures of each clusters and minimize them,
    ../../scripts/ rex
    $CHARMMEXEC nfile=[NFILE] cid=[Cluster ID] \
                < ../../scripts/cluster_ana.inp 
    [repeat for each clusters]
In the above example, "NFILE" is the number of structures in Cluster "cid". Scripts, and cluster_ana.inp can be found under directory "scripts/" once the TAR/GZ file is unzipped/untarred.

Written by Jianhan Chen (