lec13

Position restraining the ligand
-------------------------------
1. To equilibrate the protein-ligand complex, the ligand needs to be position restrained. For this, first the ligand atoms has to be indexed:

			$ gmx make_ndx -f JZ4.gro -o index_JZ4.ndx

This command is pretty clear
	- The make_ndx module of Gromacs takes in the JZ4.gro file that have been generated from the force field applied jz4_ini.pdb file.
	- As an output, it returns the index file: index_JZ4.ndx

However, there are a few more story inside the index creation, these are discussed bellow.


2. Indexing requires specifying group of atoms. After the make_ndx is called, it will show various options. The group selected is: "0 & ! a H*". Here:

			0			Stands for the system.
			! a H*		Excluding all hydrogen atoms.

Thus "0 & ! a H*" means the whole system but the hydrogen atoms should be indexed. This will generate a new option: "System_&_!H*" placed at number 3. The
previos options being:

					0	System		; 28 atoms
					1	Other		; 28 atoms
					2	JZ4			; 28 atoms

After all these are done, progress can be "save and quit" with 'q'.


3. After indexing, position restrained file for ligand is generated using the 'genrestr' module:

			$ gmx genrestr -f JZ4.gro -n index_JZ4.ndx -o posre_jz4.itp -fc 1000 1000 1000

The points to be noted about this command and the process are:
	- Position restraining parameters are always saved in files with 'itp' extension. So the output file is named "posre_jz4.itp". The '-fc' option specify
	upper limit of the restrainment - 1000 X 1000 X 1000, with 'fc' standing for "functional coordinates".
	- Rest of this command is as usual - the input file is as usual specified with '-f' option and additionally, the index file - "index_JZ4.ndx" obtained in
	the previous step is specified with the '-n' option.
	- During the execution of the command, the genrestr module will ask to specify a group. The newly generated group 3: System_&_!H have to be selected.


4. This positition restrined file posre_jz4.itp have to be added to the current topology file through an 'include' directive:

		 ...	...		...
		#include "posre.itp"
		#endif

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

As can be seen, the ligand topology file is added after the position restraining file for protein: "posre.tip". But, it can also be added after the directive
adding the ligand topology file jz4.itp.


Indexing the protein
--------------------
After the position restraining file is generated for the ligand, its time for indexing the protein:

		$ gmx make_ndx -f em.gro -o index.ndx

During the indexing, make_ndx module will ask for a group to select. The group in this occation will be "1 | 13", where no. 1 is protein and no. 13 is JZ4. This
will be labeled a new group "Protein_JZ4" and this group will be added to no. 23.


Equilibration and the MDS:
--------------------------
After this, the temperature and pressure equilibration and the final molecular dynamic simulation run is basically the same as the steps without ligand. So, the
command for these steps are listed bellow:

$ gmx grompp -f nvt.mdp -c em.gro -r em.gro -p topol.top -n index.ndx -o nvt.tpr				# Preparing the binary file for temp. equilibration.
$ gmx mdrun -deffnm nvt -v																		# MDS for temperature equilibration.
$ gmx grompp -f npt.mdp -c nvt.gro -t nvt.cpt -r nvt.gro -p topol.top -n index.ndx -o npt.tpr	# Preparing the binary file for pressure equilibration.
$ gmx mdrun -deffnm npt -v																		# MDS for pressure equilibration.
$ gmx grompp -f md.mdp -c npt.gro -t npt.cpt -p topol.top -n indx.ndx -o md1.tpr				# Preparing the binary file for final MDS.
$ gmx mdrun -deffnm md1 -v																		# The actual MD run.

There is one vital difference though, the index file is added with '-n' option with each command. Without specifying the index file like this, the equilibration
and the MDS will be done without including the ligand file. Also, the *.mdp files, that contains the specification for the MD run have to be checked and also
modified if needed.