GROMACS version: 2022

GROMACS modification: No

Hello everyone, I am trying to make free energy calculation for the first time and have a problem. I am mutating aminoacids in protein and using fast-growth method to calculate free energy of stability. Using pmx web server, I’ve created hybrid topology(which contains dummy atoms), then placed protein in a box, neutralized it with ions, and conducted energy minimization procedure.

Then after command

**gmx grompp -f nvt.mdp -c em.gro -r em.gro -p hybrid.top -o nvt.tpr -maxwarn 2**

I’ve got this warning:

The bond in molecule-type Protein_chain_A between atoms 5324 DOH and 5325

DHH has an estimated oscillational period of 6.5e-03 ps, which is less

than 5 times the time step of 2.0e-03 ps.

Maybe you forgot to change the constraints mdp option.

And then, after **gmx mdrun -deffnm nvt**

I got this error:

There are perturbed non-bonded pair interactions beyond the pair-list cutoff

of 1.111 nm, which is not supported. This can happen because the system is

unstable or because intra-molecular interactions at long distances are

excluded. If the latter is the case, you can try to increase nstlist or rlist

to avoid this.The error is likely triggered by the use of couple-intramol=no

and the maximal distance in the decoupled molecule exceeding rlist.

I have no error, if I just try to simulate this molecule without hybrid topology, so it seems that dummy particles is a source of this problem. Could please somebody help me to solve this problem. Thanks.

Here are my mdp files

**minimization.mdp**

;Run parameters

define = -DFLEXIBLE

nsteps = 1500

integrator = steep

emtol = 50.0

emstep = 0.01

;Neighbour searching

cutoff-scheme = Verlet

ns-type = grid

rcoulomb = 1.0

vdwtype = cut-off

rvdw = 1.0

nstlist = 10

rlist = 1

;Eletrostatics

coulombtype = PME

;Periodic boundary conditions

;pbc = xyz

;ld-seed = 11

; FREE ENERGY

;----------------------------------------------------

free-energy = yes

init-lambda = 0 ; start from state A

delta-lambda = 0 ; stay in state A

;

nstcomm = 100

constraints = none

coulomb-modifier = Potential-shift-Verlet

pme-order = 4

fourierspacing = 0.12

ewald-rtol = 1e-5

vdw-modifier = Potential-shift-Verlet

verlet-buffer-tolerance = 0.005

DispCorr = EnerPres

Tcoupl = no

Pcoupl = no

gen_vel = no

**nvt.mdp**

;This mdp file has been created by the pymdsetup.gromacs_wrapper.grompp.create_mdp()

;Type of MDP: nvt

;Position restrain

Define = -DPOSRES

;Run parameters

nsteps = 50000

;integrator = md

integrator = sd

dt = 0.002

comm-mode = Linear ; remove center of mass translation

;Output control

nstxout = 500

nstvout = 500

nstenergy = 500

nstlog = 500

nstcalcenergy = 100

nstcomm = 100

nstxout-compressed = 1000

compressed-x-precision = 1000

compressed-x-grps = System

;Bond parameters

constraint-algorithm = lincs

constraints = h-bonds

lincs-iter = 1

lincs-order = 4

continuation = no

;Neighbour searching

cutoff-scheme = Verlet

ns-type = grid

rcoulomb = 1.0

vdwtype = cut-off

rvdw = 1.0

nstlist = 10

rlist = 1

;Eletrostatics

coulombtype = PME

pme-order = 4

fourierspacing = 0.12

fourier-nx = 0

fourier-ny = 0

fourier-nz = 0

ewald-rtol = 1e-5

;Temperature coupling

tcoupl = V-rescale

tc-grps = Protein Non-Protein

tau-t = 0.1 0.1

ref-t = 300 300

;Pressure coupling

pcoupl = no

;Dispersion correction

DispCorr = EnerPres

;Velocity generation

gen-vel = yes

gen-temp = 300

gen-seed = -1

;Periodic boundary conditions

pbc = xyz

ld-seed = 17

; FREE ENERGY

;----------------------------------------------------

free-energy = yes

init-lambda = 0 ; start from state A

delta-lambda = 0 ; stay in state A

;

vdw-modifier = Potential-shift-Verlet