Task 8 - Molecular Dynamics Simulations

Intro

In this section we will simulate the wildtype protein and two interesting mutants with MD, e.g. the gromacs package. For this we will use an automatic pipeline. As the final simulations will take a while, we will post the analysis part at a later point.

Preparation

First we have to prepare our PDB structure. For this we will make use of repairPDB and SCWRL. Select the structure from the PDB or use your model where the missing residues have been added. A new version of repairPDB can be found in the Dropbox dir.

• use repairPDB to extract crystal water below 15 Å resolution and save into a temporary file. <-ssw 15>
• use repairPDB and extract just protein. <-nosol>
• now use SCWRL with the extracted protein and give it the PDB sequence in all small letter to complete missing sidechains
• remove the hydrogen atoms from the SCWRL output with repairPDB
• concatenate the protein and the crystal water into one file

Create important GROMACS files

Next we start to prepare the files for GROMACS:

• we have to create a TOP (topology) and a PAR (parameter) file with the following command:

pdb2gmx -f <input.pdb> -o <output.gro> -p <output.top> -water tip3p -ff amber03 -vsite hydrogens

As a model for water we choose TIP3P, as a forcefield we use AMBER03. The water is changed to virtual sites to speed up the simulation. Don't forget the file extension of the input file needs to be pdb.

• Next we create a box around the protein and fill it with water:

The following commands are used to add the box: editconf -f <file.gro> -o <file_box.gro> -bt dodecahedron -d 1.0

The -bt flag stands for boxtype; dodecahedron is a special geometrical form. -d stands for the minimum distance of the molecules to the box for any surface residue in nanometers.

genbox -cp <file_box.gro> -cs spc216.gro -p <file.top> -o <file_water.gro>

cs tells the program which parameters/topology to use for the water - in this case the ones that are compatible with tip3p

• In the next step we have to add the ions. To do so we need to used the grompp command, which is always used to prepare a system for more complicated steps. This command needs an mdp file where we can define several additional parameters.

The file will include the following info and should have the file extension .mdp:

integrator = steep
emtol = 1.0
nsteps = 500
nstenergy = 1
energygrps = System
coulombtype = PME
rcoulomb = 0.9
rvdw	 = 0.9
rlist = 0.9
fourierspacing = 0.12
pme_order = 4
ewald_rtol = 1e-5

For details of the settings please check the gromacs manual, e.g. found in the dropbox literature folder.

• We call grommp the following way:

grompp -v -f <file.mdp> -c <file_water.gro> -p <file.top> -o <file_water.tpr>

The .tpr file is what we need in the next step where we use the command genion.

• Genion will add the solvent to the system and neutralize the charge that is part of the system due to charged amino acids.

genion -s <file_water.tpr> -o <file_solv.pdb> -conc 0.1 -neutral -pname NA+ -nname CL-

As the selection we will use number 13, which means that we replace the water molecules with the ions. -conc defines the concentration of the ions which is 0.1 molar. -neutral tells the command to neutralize the charges. -pname is the name of the positively charged ions to add, -nname the negatively charged.

• Now we will have to do some manual work. From the output of the last command you should note how many CL- and NA+ ions have been added and how many solvent molecules are now given. These values need to be changed in the top file; copy the original and edit the copy. At the end of the file there are entries that begin with SOL. From the larger one you subtract the number of ions added. Then add the entry NA and the number of added NA+ ions and the entry CL and the number of added CL- ions.

• To make sure our initial crystal water does not overlap with the added water we use repairPDB again.

repairPDB <file_solv.pdb> -cleansol <file_solv2.pdb>

In the last line there is a REMARK tag with the number of removed water molecules. This has to be changed in the TOP file if larger than 0.

• Before we are able to start with the minimization procedures we need to extract restraints from the structures. These restraints are useful to reduce the simulation time by disallowing very fast vibrations such as seen for hydrogen atoms. The command we use is called genrestr.

genrestr -f <file_solv2.pdb> -o <file.itp>

In the command line menu we choose number 1 for protein.

Minimization solvent

Now we can start our first minimization - minimizing the solvent only and restraining the protein.

• To prepare the system we once again need to use grompp and therefore create a mdp file.

The MDP file looks like this:

define = -DPOSRES
integrator = steep
emtol = 1.0
nsteps = 500
nstenergy = 1
energygrps = System
coulombtype = PME
rcoulomb	 = 0.9
rvdw = 0.9
rlist = 0.9
fourierspacing = 0.12
pme_order = 4
ewald_rtol = 1e-5
pbc = xyz

• We call grompp the following way:

grompp -v -f <file.mdp> -c <file_solv2.pdb> -p <file.top> -o <file_solv_min.tpr>

• Now we do our first run with the simulation command mdrun.

If you have multiple processors you do the following:

mpirun -np <number of processors to use> mdrun_mpi -v -deffnm <file_solv_min> -c <file_solv_min.pdb>

If you only run it on one processor it looks like this:

mdrun -v -deffnm <file_solv_min> -c <file_solv_min.pdb>

Minimization system

Now we have a file where the solvent has been minimized.

• Next we need to minimize the solvent and the protein sidechains.

We have to recreate the position restraints files. Choose option 4 which only restraints the backbone of the protein.

genrestr -f <file_solv_min.pdb> -o <file.itp>

• The mdp file from the previous minimization can be reused; therefore, we only have to use grompp to prepare our tpr files.

grompp -v -f <file.mdp> -c <file_solv_min.pdb> -p <file.top> -o <file_solv_min2.tpr>

• If you have multiple processors you do the following:

mpirun -np <number of processors to use> mdrun_mpi -v -deffnm <file_solv_min2> -c <file_solv_min2.pdb>

• If you only run it on one processor it looks like this:

mdrun -v -deffnm <file_solv_min2> -c <file_solv_min2.pdb>

Equilibration of system

Now that our system is minimized we do have to start equilibrating it. The following procedures will take a while but should still be calculated locally and not using the LRZ account.

• In the next step we will apply temperature coupling. In this step we will slowly heat up our system to the wished temperature. In this simulation we have a constant particle number, volume and finally temperature.

You will have to create a new mdp file called nvt.mdp

define = -DPOSRES
integrator = md
dt = 0.005 
nsteps = 10000
nstxout = 0
nstvout = 0
nstfout = 0
nstlog = 1000
nstxtcout	 = 0
nstenergy = 5
energygrps = Protein Non-Protein
nstcalcenergy = 5
nstlist = 10
ns-type = Grid
pbc = xyz
rlist = 0.9
coulombtype = PME
rcoulomb = 0.9
rvdw = 0.9
fourierspacing = 0.12
pme_order = 4
ewald_rtol = 1e-5      
gen_vel = yes   
gen_temp = 200.0 
gen_seed = 9999  
constraints = all-bonds
tcoupl = V-rescale		
tc-grps = Protein  Non-Protein 
tau_t = 0.1	0.1 	
ref_t = 298	298 
pcoupl = no

For details regarding each setting check the gromacs manual.

• We use once again grompp to prepare the tpr file for mdrun.

grompp -v -f <nvt.mdp> -c <file_solv_min2.pdb> -p <file.top> -o <file_nvt.tpr>

• Then we call mdrun as above without the -c option:

... mdrun -v -deffnm file_nvt

• Next we create an .mdp file for pressure coupling NPT.

define = -DPOSRES
integrator = md
dt = 0.005 
nsteps = 10000
nstxout = 0
nstvout = 0
nstfout = 0
nstlog = 1000
nstxtcout = 0
nstenergy = 5
xtc_precision = 1000
xtc-grps = System
energygrps = Protein Non-Protein
nstcalcenergy = 5
nstlist = 5
ns-type = Grid
pbc = xyz
rlist = 0.9
coulombtype = PME
rcoulomb = 0.9
rvdw = 0.9
fourierspacing = 0.12
pme_order = 4
ewald_rtol = 1e-5
tcoupl = V-rescale 
tc-grps = Protein Non-Protein 
tau_t = 0.1 0.1 
ref_t = 298 298 
pcoupl = Berendsen
Pcoupltype = Isotropic
tau_p = 1.0
compressibility = 4.5e-5
ref_p = 1.0
gen_vel = no
constraints = all-bonds

• We use once again grompp to prepare the tpr file for mdrun.

grompp -v -f <npt.mdp> -c <file_nvt.gro> -p <file.top> -o <file_npt.tpr>

• Then we call mdrun as above:

... mdrun -v -deffnm file_npt

Production run

Finally, we come to the production run. Again we do everything as above. Only the final step, the MD simulation we will submit to the LRZ linux cluster. Below I will suggest one possible way to do this - of course you can do this in a different way. Also, be aware that this might take a while, as the queues are generally really full.

• This is our md.mdp file:

integrator = md
tinit = 0
dt = 0.005
nsteps = 2000000
nstxout = 50000
nstvout = 50000
nstfout = 0
nstlog = 1000
nstxtcout = 1000
nstenergy = 1000
energygrps = Protein Non-Protein 
nstcalcenergy = 5
nstlist = 5
ns-type = Grid
pbc = xyz
rlist = 0.9
coulombtype = PME
rcoulomb = 0.9
rvdw = 0.9
fourierspacing = 0.12
pme_order = 4
ewald_rtol = 1e-5
tcoupl = V-rescale 
tc-grps = Protein Non-Protein 
tau_t = 0.1 0.1 
ref_t = 298 298 
pcoupl = Berendsen
Pcoupltype = Isotropic
tau_p = 2.0
compressibility = 4.5e-5
ref_p = 1.0
gen_vel = no
constraints = all-bonds
constraint-algorithm = Lincs
unconstrained-start = yes
lincs-order = 4
lincs-iter = 1
lincs-warnangle = 30
comm_mode = linear

• We use once again grompp to prepare the .tpr file for mdrun.

grompp -v -f <md.mdp> -c <file_npt.gro> -p <file.top> -o <file_md.tpr>

Try the mdrun locally, just to see whether all is ok:

... mdrun -v -deffnm file_md

LRZ

From now on we have to deal with the LRZ.

• First we have to copy our files to the system. This can be achieved using scp. You need the .mdp, .gro, .top and .tpr files used/produced in the last grompp call.

To login to the LRZ use the following nodes: ssh -XY username@lx64ia2.lrz.de

or

ssh -XY username@lx64ia3.lrz.de

More info at http://www.lrz.de/services/compute/linux-cluster/batch/#batchSGE

• There you have to create a configuration file (bla.cmd):

#!/bin/bash
#$-o $HOME/DIR/mdrun.WT_1.out -j y
#$-N PROJECTNAME(no number at beginning)
#$-S /bin/bash
#$-M YOUR@EMAIL.COM
#$-l h_rt=32:00:00
#$-l march=x86_64
#$-pe mpi_32 32
. /etc/profile
cd $HOME/DIR
$HOME/DIR/mpirun -np 32 mdrun_mpi -v -deffnm $HOME/DIR/file_md

• Edit the ~/.bashrc and add this to the end in order to load gromacs:

module load gromacs/4.6

• Finally to submit the job type qsub bla.cmd

• To check if everything is ok type qstat and look for your job...

HAVE FUN !!!