Hello everyone, I am doing free energy calculations upon aminoacid mutation for the first time and got a problems. I want to calculate equilibrium trajectories for fast-growth method.

Here is what I did.

- Downloaded PDB structure from RCSB
- Splitted it into several chains(because I dont know how to rebuild missing residues in in multimeric protein)
- Rebuilt missing residues in every chain using Modeller
- Combined all chains together
- Created hybrid topology using PMX
- Added box, water and ions in Gromacs

During minimization step there was 1 atom that Gromacs tried to minimize 90% of time.

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

I’ve got 2 warnings

WARNING 1 [file hybrid_posre_Protein_chain_A.itp, line 2692]:

Some parameters for bonded interaction involving perturbed atoms are

specified explicitly in state A, but not B - copying A to B

WARNING 2 [file hybrid.top, line 19]:

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**

Fatal 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.

Could you please help me to solve this issue. It seems for me(maybe I am wrong) that the source of the problem are dummy atoms, because without hybrid topology(without dummy atoms) I dont have any problems. Maybe dummy atoms make clashes with another residues. If so, is there right way how to construct hybrid topology without clashes? 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

; 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**

;Type of MDP: nvt

;Position restrain

Define = -DPOSRES

;Run parameters

nsteps = 50000

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