Molecular Dynamics Simulations

Preparation

• We extracted all crystal water by repairPDB 1a6z.pdb -ssw 15t >ssw.tmp but, there was no crystal water included.
• Extracting the pure protein by repairPDB 1a6z.pdb -nosol >1a6z_clean.pdb
• Complete missing side chains with scwrl -i 1a6z_clean.pdb -s 1a6z_low.txt -o 1a6z_clean_complete.pdb > out.log
• Remove all H-Atoms by repairPDB 1a6z_clean_complete.pdb -noh >1a6z_final.pdb
• because there was no crystal water in the origin pdb included, we had nothing to concatenate.

Creation of the gromac files

• pdb2gmx -f 1a6z_final.pdb -o 1a6z_final.gro -p 1a6z_final.top -water tip3p -ff amber03 -vsite hydrogens

Then a box around our protein was added by editconf -f 1a6z_final.gro -o 1a6z_final_box.gro -bt dodecahedron -d 1.0
and filled this box with water by using genbox -cp 1a6z_final_box.gro -cs spc216.gro -p 1a6z_final.top -o 1a6z_final_box_water.gro

Now we create the .tpr file for further use with genion by grompp -v -f first.mdp -c 1a6z_final_box_water.gro -p 1a6z_final.top -o 1a6z_final_box_water.tpr

Genion is used with genion -s 1a6z_final_box_water.tpr -o 1a6z_solv.pdb -conc 0.1 -neutral -pname NA+ -nname CL- Now we counted the added ions by using tr -s ' ' '\n' < out.txt | grep -c 'ION'. ION is set to NA+ and CL- to get the number of added ions. For CL- genion added 41 and for NA+ genion added 52 ions.

• the Next step was to edit the SOL entry in the .top file. Here we found a SOL entry of 21363 which we changed to 21363-52-41=21270 and added two new entrys, NA with the value 52 and CL with the value 41.

• After adding the new water, we checked for an overlab with the crystal waterby using repairPDB 1a6z_solv.pdb -cleansol > 1a6z_solv2.pdb, but as we had no crystal water in our file, we assumed to have no overlab. And as we found no REMARK tag in the new file, we should have no overlab.

• In this step we added restrains to disable very fast vibrations such as seen for hydrogen atoms to reduce the simulation time. The command we used was genrestr -f 1a6z_solv2.pdb -o 1a6z.itp

Minimization solvent and system

• We first created a new .tpr file with grompp -v -f second.mdp -c 1a6z_solv.pdb -p 1a6z_final_ion.top -o 1a6z_solv_min.tpr. But if we used the pdb-file created with the last repairPDB call which should check for overlabing water, we get an error which tells us that the number of coordinates in the pdb file does not match the number of coordinates in the top-file. Therefore we used the pdb-file which was not "repaired".
• To minimise the solvend we used the command mpirun -np 4 mdrun_mpi -v -deffnm 1a6z_solv_min.tpr -c 1a6z_solv_min.pdb.
• To minimise the solvent and the protein sidechains we used the commands genrestr -f 1a6z_solv_min.pdb -o 1a6z_min.itp to create a new .itp-file. grompp -v -f second.mdp -c 1a6z_solv_min.pdb -p 1a6z_final_ion.top -o 1a6z_solv_min2.tpr to prepare a new .tpr file. With the last command mpirun -np 4 mdrun_mpi -v -deffnm 1a6z_solv_min2.tpr -c 1a6z_solv_min2.pdb we minimised the system.

Equilibration of system

• First we created a new .tpr file using grompp -v -f <nvt.mdp> -c <file_solv_min2.pdb> -p <file.top> -o <file_nvt.tpr> and then started rhe mdrun with mpirun -np 4 mdrun_mpi -v -deffnm 1a6z_nvt.tpr -c 1a6z_nvt.pdb.
• The second step was to create a new .tpr file for pressure coupling by grompp -v -f <npt.mdp> -c <file_nvt.gro> -p <file.top> -o <file_npt.tpr>. But to get an adepted .gro file, we had to use pdb2gmx a second time with pdb2gmx -f <input.pdb> -o <output.gro> -p <output.top> -water tip3p -ff amber03 -vsite hydrogens. For startinf the mdrun we used again the command mpirun -np 4 mdrun_mpi -v -deffnm 1a6z_nvt.tpr -c 1a6z_nvt.pdb