Task 8: Molecular Dynamics Simulations
- 1 Task description
- 2 Intro and selection of two mutants
- 3 Preperation
- 4 Create important GROMACS files
- 5 Minimization solvent
- 6 Minimization system
- 7 Equilibration of system
- 8 Production run
- 9 LRZ
A detailed task description can be found here.
Intro and selection of two mutants
The description given here was applied for our wild type (1J8U). However, the steps done for our two mutants R408W and P281L are the same.
We selected R408 we because it is the most abundant mutation of PAH which is associated with phenylketonuria. It has a total frequency of 6.67% (see ).
The second mutation, P281L, was selected because it is the closed mutation to the binding site (HIS 285) and we hope that we are able to see some interesting things here which give us an explanation why this mutation is disease causing.
Before we can start to generate all necessary files for our MD simulation we have to prepare our structure first. We employed the following steps:
1. Extracting the crystal water below 15 Å
repairPDB 1J8U.pdb -ssw 15 > water_below_15.out
We received the following output:
HETATM 2559 O HOH A1008 2.996 9.738 16.094 1.00 14.91 O HETATM 2561 O HOH A1010 -3.667 12.788 8.538 1.00 14.80 O HETATM 2572 O HOH A1021 3.557 27.911 10.913 1.00 14.69 O HETATM 2879 O HOH A1328 -5.335 24.271 14.490 1.00 14.88 O TER
2. Extract only the protein
repairPDB 1J8U.pdb -nosol > 1J8U_nosol.pdb
3. Extract the amino acid sequence and turn it into lower case letter to give it as a input for SCWRL
repairPDB 1J8U.pdb -seq > 1J8U_seq.txt
vim 1J8U_seq.txt :%s/.*/\L&/g
4. Use SCWRL to complete the side chains of all amino acids
/apps/scwrl4/Scwrl4 -i 1J8U_nosol.pdb -s 1J8U_seq.txt -o 1J8U_nosol_after_SCWRL.pdb | tee scwrl_1J8U_nosol_after_scwrl.out
5. Remove the hydrogen atoms from the SCWRL output
repairPDB 1J8U_nosol_after_SCWRL.pdb -noh > 1J8U_nosol_after_SCWRL_no_h.pdb
6. concatenate the protein and the crystal water into one file
We just added the output of step 1 to the end of the PDB file 1J8U_nosol_after_SCWRL_no_h.pdb and named this file 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.pdb
Create important GROMACS files
1. We have to create a TOP (topology) and a PAR (parameter) file with the following command:
pdb2gmx -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.pdb -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.gro -p 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.top -water tip3p -ff amber03 -vsite hydrogens | tee pdb2gmx.out
2. Next we create a box around the protein and fill it with water
Create the box:
editconf -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.gro -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_box.gro -bt dodecahedron -d 1.0 | tee editconf.out
Fill it with water:
genbox -cp 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_box.gro -cs spc216.gro -p 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.top -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_water.gro
3. In the next step we have to add the ions. To do so we need to use 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.
Create parameter file addions.mdp:
And fill it with the following content:
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
Now, we execute it with this command:
grompp -v -f addions.mdp -c 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_water.gro -p 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.top -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_water.tpr | tee grommp.out
4. Now we add the solvent to the system and neutralize the charge that is part of the system due to charged amino acids. To do so we use genion:
genion -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_water.tpr -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv.pdb -conc 0.1 -neutral -pname NA+ -nname CL- | tee genion.out
During the call we were asked for a number we selected 13.
5. Next we have to adjust the SOL value and add the number of NA+ and CL- in our 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.top
We had 25 NA+ and 21 CL- ions generated from our last step. So we had to reduce the number in the SOL field to 10170 - 46 = 10124 the .top file had in the end the following values:
[ molecules ] ; Compound #mols Protein_chain_A 1 SOL 4 SOL 10124 NA 25 CL 21
6. To make sure our initial crystal water does not overlap with the added water we use repairPDB again.
repairPDB 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv.pdb -cleansol > 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2.pdb
7. 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.
REMARK RM 0
Value is 0 so we did not have to change anything.
8. 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 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2.pdb -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2.itp | tee genrestr.out
In the command line menu we choose number 1 for protein.
1. Creating a .mdp file for grompp to prepare the system for the solvent minimization
We created an file with the name minimize_solvent.mdp by using vim:
The content of the file is as follows:
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
2. execution of grompp to prepare the system for the MD run
grompp -v -f minimize_solvent.mdp -c 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2.pdb -p 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.top -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2_min.tpr | tee grompp_minimize_solvent.out
3. Executing mdrun to minimize the solvent
mdrun -v -deffnm 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2_min -c 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2_min.pdb | tee mdrun_minimize_solvent.out
In this part we minimize the solvent AND the side chains of the protein.
1. Creating the position restraint file.
genrestr -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2_min.pdb -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2_min.itp | tee genrestr_minimize_system.out
We selected option 4 which says that we only put restraints on the backbone of the protein.
2. Create an .mdp file for the grompp call
We use the same .mdp from the solvent minimization step
3. Calling grompp to prepare the tpr file for our mdrun step
grompp -v -f minimize_solvent.mdp -c 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2_min.pdb -p 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.top -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2_min2.tpr | tee grompp_minimize_system.out
4. Calling mdrun to minimize the system
mdrun -v -deffnm 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2_min2 -c 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2_min2.pdb | tee mdrun_minimize_system.out
Equilibration of system
Heating up the System with constant volume and particle number (NVT)
Here we will heat up our system to a certain temperature. We will keep our particle number and volume constant, the pressure is changing.
1. creating a .mdp configuration file for grompp
We created a mdp file with the name nvt.mdp :
The content is as follows:
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
2. Creating .tpr file for our MD run by employing grompp
grompp -v -f nvt.mdp -c 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_solv2_min2.pdb -p 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.top -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_nvt.tpr | tee grompp_equi_system_nvt.out
3. MD run
mdrun -v -deffnm 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_nvt | tee mdrun_equi_system_nvt.out
Heating up the System with constant pressure and particle number (NPT)
Here we will heat up our system to a certain temperature. We will keep our particle number and pressure constant, the volume is changing.
1. Creating an .mdp file for pressure coupling NPT
We create a .mdp file with the following command:
And the content:
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
2. Creating .tpr file for our MD run by employing grompp
grompp -v -f npt.mdp -c 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_nvt.gro -p 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.top -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_npt.tpr | tee grompp_equi_system_npt.out
3. MD run
mdrun -v -deffnm 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_npt | tee mdrun_equi_system_npt.out
In this part we prepare the files for our final production run on the LRZ cluster.
1. Creating a .mdp parameter file for grompp
We created a file with the following command:
And this content:
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
2. Creating a .tpr file for our MD run
grompp -v -f md.mdp -c 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_npt.gro -p 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water.top -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr | tee grompp_prduction_run_md.out