I have done md simulation using following commands-

what is the meaning of this warning, it it harmful, hI attached e warning image and nvt.mdp in which I have done few changes

gmx

Add Amber99SB-ILDN forcefield

gmx pdb2gmx -ff amber99sb-ildn -f target.pdb -o DNA.pdb -p topol.top -water spce -ignh

Add hydrogen to Ligand.pdb using Pymol

antechamber -i Ligand.pdb -o Ligand.mol2 -fi pdb -fo mol2 -c gas

copy acpype.py from acpype_lib in your folder

python acpype.py -i Ligand.mol2

# Merge DNA.pdb + updated Ligand_NEW.pdb -> Complex.pdb

grep -h ATOM DNA.pdb Ligand.acpype/Ligand_NEW.pdb >| Complex.pdb

# Edit topol.top -> Complex.top

cp Ligand.acpype/Ligand_GMX.itp Ligand.itp

cp topol.top Complex.top

# `#include "Ligand.itp"` has to be inserted right after

# `#include "amber99sb.ff/forcefield.itp"`

# line and before `Protein_*.itp` line in _Complex.top_

cat Complex.top | sed '/forcefield\.itp\"/a\

#include "Ligand.itp"

' >| Complex2.top

echo "Ligand 1" >> Complex2.top

mv Complex2.top Complex.top

#newbox is formed

gmx editconf -bt triclinic -f Complex.pdb -o Complex.pdb -d 1.5

#solvate this box

gmx solvate -cp Complex.pdb -cs spc216.gro -o Complex_b4ion.pdb -p Complex.top

#download ions.mdp from tutorial of protein ligand complex of Gromacs

gmx grompp -f ions.mdp -c Complex_b4ion.pdb -p Complex.top -o Complex_b4ion.tpr

cp Complex.top Complex_ion.top

echo 6| gmx genion -s Complex_b4ion.tpr -o Complex_b4em.pdb -neutral -conc 0.15 -p Complex_ion.top

#grp no. is acc to SOL

Select a continuous group of solvent molecules

Group 0 ( System) has 15639 elements

Group 1 ( DNA) has 478 elements

Group 2 ( UNK) has 59 elements

Group 3 ( Other) has 59 elements

Group 4 ( UNK) has 59 elements

Group 5 ( Water) has 15102 elements

Group 6 ( SOL) has 15102 elements

Group 7 ( non-Water) has 537 elements

Select a group: Selected 6: 'SOL'

Number of (3-atomic) solvent molecules: 5034

mv Complex_ion.top Complex.top

#download em.mdp from tutorial of protein ligand complex of Gromacs

gmx grompp -f em.mdp -c Complex_b4em.pdb -p Complex.top -o em.tpr

gmx mdrun -v -deffnm em

#Restraining the Ligand

gmx make_ndx -f Ligand.pdb -o index_Ligand.ndx

> 0 & ! a H*

> q

gmx genrestr -f Ligand.pdb -n index_Ligand.ndx -o posre_Ligand.itp -fc 1000 1000 1000

Select group to position restrain

Group 0 ( System) has 59 elements

Group 1 ( Other) has 59 elements

Group 2 ( UNK) has 59 elements

Group 3 ( System_&_!H*) has 25 elements

Select a group: 3

Selected 3: 'System_&_!H*'

#include posre_Ligand.itp in topology file i.e. Complex.top in this manner

; Include Position restraint file

#ifdef POSRES

#include "posre.itp"

#endif

; Include ligand topology

#include "Ligand.itp"

; Ligand position restraints

#ifdef POSRES

#include "posre_Ligand.itp"

#endif

; Include water topology

#include "./charmm36-mar2019.ff/tip3p.itp"

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

0 System : 15551 atoms

1 DNA : 478 atoms

2 UNK : 59 atoms

3 NA : 29 atoms

4 CL : 15 atoms

5 Other : 59 atoms

6 UNK : 59 atoms

7 NA : 29 atoms

8 CL : 15 atoms

9 Water : 14970 atoms

10 SOL : 14970 atoms

11 non-Water : 581 atoms

12 Ion : 44 atoms

13 UNK : 59 atoms

14 NA : 29 atoms

15 CL : 15 atoms

16 Water_and_ions : 15014 atoms

nr : group '!': not 'name' nr name 'splitch' nr Enter: list groups

'a': atom '&': and 'del' nr 'splitres' nr 'l': list residues

't': atom type '|': or 'keep' nr 'splitat' nr 'h': help

'r': residue 'res' nr 'chain' char

"name": group 'case': case sensitive 'q': save and quit

'ri': residue index

Merge the "DNA" and "UNK" groups with the following, where ">" indicates the make_ndx prompt:

> 1|2

Copied index group 1 'DNA'

Copied index group 2 'UNK'

Merged two groups with OR: 478 59 -> 537

17 DNA_UNK : 537 atoms

> q

#We can now set tc-grps = DNA_UNK Water_and_ions in nvt.mdp, npt.mdp and md.mdp

#download nvt.mdp from tutorial of protein ligand complex of Gromacs

gmx grompp -f nvt.mdp -c em.gro -r em.gro -p Complex.top -n index.ndx -o nvt.tpr

gmx mdrun -deffnm nvt