How to apply position restraint on crystal water?

GROMACS version: 2018.8

Hi, everyone!

I am working on a protein-ligand complex system, I wanna save 2 water molecules in the ligand binding pocket and apply position restraint for O atom. According to some discussion online, firstly I use pdb2gmx to prepare the protein and crystal water’s topology file (named 4oa4_processed.gro), then I copy the ‘tip3p.itp’ file and give the file a new name ‘tip3p1.tip’ , fill in a new name ‘FIX’ for the [ molecule_type ] in this .itp file, and also delete the [ settles ] section. Then, I include the tip3p1.tip and ligand .itp file in my .top file and change the ‘SOL’ to ‘FIX’. Details are showed in the attached .top file. When I use grompp to add ions, notes occurs as follows:

‘’
NOTE 3 [file topol.top, line 45825]:
In moleculetype ‘FIX’ 3 atoms are not bound by a potential or constraint
to any other atom in the same moleculetype. Although technically this
might not cause issues in a simulation, this often means that the user
forgot to add a bond/potential/constraint or put multiple molecules in
the same moleculetype definition by mistake. Run with -v to get
information for each atom.
‘’

It seems that it failed to add restraint to FIX, namely the crystal water. I don’t know how to solve it.

----------------------------------------------------the .top file-------------------------------------------
; Include Position restraint file
#ifdef POSRES
#include “posre.itp”
#endif

; Include ligand topology
#include “sin.itp”

; Include fixed water topology
#include “./charmm36-feb2021.ff/tip3p1.itp”

#ifdef POSRES
; Position restraint for each water oxygen
[ position_restraints ]
; i funct fcx fcy fcz
1 1 1000 1000 1000
#endif

; Include water topology
#include “./charmm36-feb2021.ff/tip3p.itp”

#ifdef POSRES_WATER
; Position restraint for each water oxygen
[ position_restraints ]
; i funct fcx fcy fcz
1 1 1000 1000 1000
#endif

; Include topology for ions
#include “./charmm36-feb2021.ff/ions.itp”

[ system ]
; Name
Protein in water

[ molecules ]
; Compound #mols
Protein_chain_A 1
sin 1
FIX 2
SOL 18462


--------------------------------------the tip3p1.tip------------------------------------------------------

[ moleculetype ]
; molname nrexcl
FIX 2

[ atoms ]
; id at type res nr residu name at name cg nr charge
#ifdef _FF_CHARMM
1 OT 1 FIX OW 1 -0.834
2 HT 1 FIX HW1 1 0.417
3 HT 1 FIX HW2 1 0.417
#endif

#ifdef FLEXIBLE

#ifdef ORIGINAL_TIP3P
[ bonds ]
; i j funct length force.c.
1 2 1 0.09572 502416.0 0.09572 502416.0
1 3 1 0.09572 502416.0 0.09572 502416.0

[ angles ]
; i j k funct angle force.c.
2 1 3 1 104.52 628.02 104.52 628.02

#else
;CHARMM TIP3p
[ bonds ]
; i j funct length force.c.
1 2 1 0.09572 376560.0 0.09572 376560.0
1 3 1 0.09572 376560.0 0.09572 376560.0

[ angles ]
; i j k funct angle force.c.
2 1 3 1 104.52 460.24 104.52 460.24
#endif

#else
[ exclusions ]
1 2 3
2 1 3
3 1 2
#endif


The second question is that how to apply position restraint for FIX’s O atom during NVT/NPT equilibrium phase. I think I need to use ‘define = -DPOSRES’ and ‘tc-grps = Protein_sin_FIX Water_and_ions’, the ‘Protein_sin_FIX’ stands for the group made by protein, ligand and crystal water. And when running run production MD, I need to release the restraints by not using ‘define = -DPOSRES’ and using ‘tc-grps = Protein_sin_FIX Water_and_ions’. Is it right?

This problem has bothered me for one week. Any suggestion would be of great help to me. Thanks a lot!

Followed Mr. Jalemkul's advice in this post [https://gromacs.bioexcel.eu/t/solvent-partition/1034](https://gromacs.bioexcel.eu/t/solvent-partition/1034), after deleting the [ settles ] section, I use a [ constraints ] block as follows, then the Note above disappeared.

[ constraints ]
1 2 1 0.09572
1 3 1 0.09572
2 3 1 0.15139

please see kalp15 in dppc tutorial by Dr. Justin in the topology preparation section you will see how to position restrain specific atoms of your system. Translate trick for your case.

Other way can be to use charmmgui web server retaining crystal water in input file. Get the system from there
Before minimizing prepare separate restraints for water molecules of interest in gromacs.
and add the restrain file to your .mdp
I hope it helps.

OK, thanks a lot.