# Free energy calculations error

**URL:** <https://gromacs.bioexcel.eu/t/free-energy-calculations-error/4005>\
**Category:** User discussions\
**Tags:** pmx\
**Created:** [April 12, 2022, 3:52pm UTC](https://gromacs.bioexcel.eu/t/free-energy-calculations-error/4005 "2022-04-12T15:52:43Z")\
**Posts on this page:** 2\
**Page:** 1

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**Author:** ![cyber222](https://avatars.discourse-cdn.com/v4/letter/c/3be4f8/32.png) [@cyber222](https://gromacs.bioexcel.eu/u/cyber222)\
**Post date:** [April 12, 2022, 3:52pm UTC](https://gromacs.bioexcel.eu/t/free-energy-calculations-error/4005/1 "2022-04-12T15:52:43Z")

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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](http://pmx.mpibpc.mpg.de/dna_webserver.html), 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

---

<div class="post-metadata">

**Author:** ![pinoji1](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/pinoji1/32/1442_2.png) [@pinoji1](https://gromacs.bioexcel.eu/u/pinoji1)\
**Post date:** [November 1, 2023, 12:05am UTC](https://gromacs.bioexcel.eu/t/free-energy-calculations-error/4005/2 "2023-11-01T00:05:22Z")

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This maybe too late, but add -nt 16 on mdrun command.