Equilibrating our protein-ligand complex will be much like equilibrating any other system containing a protein in water. There are a few special considerations, in this case:

1. Applying restraints to the ligand
2. Treatment of temperature coupling groups

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.

Thermostats

Proper control of temperature coupling is a sensitive issue. Coupling every moleculetype to its own thermostatting group is a bad idea, though it may seem like this approach may be more accurate. For instance, if you do the following:

tc-grps = Protein JZ4 SOL CL

your system will probably blow up, since the temperature coupling algorithms are not stable enough to control the fluctuations in kinetic energy that groups with a few atoms (i.e., JZ4 and CL) will produce. Do not couple every single species in your system separately.

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