Equilibrating our protein-ligand complex will be much like equilibrating any other system containing a protein in water. We need to consider one additional attribute, which is how to apply position restraints to the JZ4 ligand.

Restraining the Ligand

To restrain the ligand, we will need to generate a position restraint topology for it. First, create an index group for JZ4 that contains only its non-hydrogen atoms:

gmx make_ndx -f jz4.gro -o index_jz4.ndx
...
 > 0 & ! a H*
 > q

Then, execute the genrestr module and select this newly created index group (which will be group 3 in the index_jz4.ndx file):

gmx genrestr -f jz4.gro -n index_jz4.ndx -o posre_jz4.itp -fc 1000 1000 1000

The previous edits to the ligand topology prepared us for this step, such that we now have the "posre_jz4.itp" file that allows us to restrain the ligand when position restraints are invoked.

If you want a bit more control during equilibration, i.e. restraining the protein and ligand independently, you could instead control the inclusion of the ligand position restraint file in a different #ifdef block, like so:

; Ligand position restraints
#ifdef POSRES_LIG
#include "posre_jz4.itp"
#endif

In the latter case, to restrain both the protein and the ligand, we would need to specify define = -DPOSRES -DPOSRES_LIG in the .mdp file. How you want to treat your system is up to you. These examples are meant only to illustrate the flexibility GROMACS provides. For a standard equilibration procedure, restraining the protein and ligand simultaneously is probably sufficient. Your own needs may vary.

Equilibration

Proceed with NVT equilibration using this .mdp file.

gmx grompp -f nvt.mdp -c em.gro -r em.gro -p topol.top -o nvt.tpr

gmx mdrun -deffnm nvt

Once the NVT simulation is complete, proceed to NPT with this .mdp file:

gmx grompp -f npt.mdp -c nvt.gro -t nvt.cpt -r nvt.gro -p topol.top -o npt.tpr

gmx mdrun -deffnm npt