# Perturbed non-bonded pair interaction beyond the pair list cutoff **URL:** \ **Category:** User discussions\ **Tags:** mdp-parameters\ **Created:** [August 12, 2022, 12:49am UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596 "2022-08-12T00:49:25Z")\ **Posts on this page:** 13\ **Page:** 1
**Author:** ![kyrstyno](https://avatars.discourse-cdn.com/v4/letter/k/d07c76/32.png) [@kyrstyno](https://gromacs.bioexcel.eu/u/kyrstyno)\ **Post date:** [August 12, 2022, 12:49am UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/1 "2022-08-12T00:49:25Z")
GROMACS version: 2021.3 GROMACS modification: No I have been trying to minimize the energy of a coarse-grained MARTINI ion channel- membrane system that I constructed with the CHARMM membrane GUI builder. I keep running into the following error even though I have increased rvdw/rcoulomb from 1.1 to 2 and increased nstlist from 20 to 40. I even tried increasing rvdw/rcoulomb from 1.1 to 5 and nstlist to 50, and I ended up with the same error message even though the system energy minimized to step 1103 with a potential energy of -5.48e6. I have read the other issues on this forum with error messages similar to this one, but it wasn’t clear to me what the solution is or what I should do to move past this error. Thank you for any guidance! Error message: Energies (kJ/mol) Bond G96Angle Proper Dih. Improper Dih. LJ (SR) 4.12162e+05 3.54244e+04 2.61001e+03 7.16265e+03 -5.90651e+06 Coulomb (SR) Potential Pressure (bar) dVremain/dl Constr. rmsd -1.96625e+04 -5.46881e+06 4.37849e+02 5.42968e+06 3.21331e-06 ``` Step Time 250 250.00000 ``` Energies (kJ/mol) Bond G96Angle Proper Dih. Improper Dih. LJ (SR) 4.13072e+05 3.54125e+04 2.61052e+03 7.15756e+03 -5.90886e+06 Coulomb (SR) Potential Pressure (bar) dVremain/dl Constr. rmsd -1.96655e+04 -5.47028e+06 4.31277e+02 5.43278e+06 3.28816e-06 * * * Program: gmx mdrun, version 2021.3 Source file: src/gromacs/gmxlib/nonbonded/nb\_free\_energy.cpp (line 854) Fatal error: There are 1 perturbed non-bonded pair interactions beyond the pair-list cutoff of 2.1 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. ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [August 12, 2022, 2:17am UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/2 "2022-08-12T02:17:21Z")
Does the simulation run when you’re not performing any kind of perturbation? It seems perhaps your system simply isn’t well equilibrated enough to start making transformations. Minimize and equilibrate using normal MD methods, then apply the free energy features. ---
**Author:** ![kyrstyno](https://avatars.discourse-cdn.com/v4/letter/k/d07c76/32.png) [@kyrstyno](https://gromacs.bioexcel.eu/u/kyrstyno)\ **Post date:** [August 12, 2022, 2:31am UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/3 "2022-08-12T02:31:33Z")
In that case do you recommend turning off the free energy? I am not performing perturbations on the system yet. mdp file: tinit = 0.0 nsteps = 5000 nstlog = 100 nstenergy = 100 nstxout-compressed = 1000 compressed-x-precision = 100 cutoff-scheme = Verlet nstlist = 20 ns\_type = grid pbc = xyz verlet-buffer-tolerance = 0.005 epsilon\_r = 15 coulombtype = reaction-field rcoulomb = 5 vdw\_type = cutoff vdw-modifier = Potential-shift-verlet rvdw = 5 tcoupl = v-rescale tc-grps = protein membrane solute tau\_t = 1.0 1.0 1.0 ref\_t = 323.15 323.15 323.15 ; Pressure coupling: Pcoupl = berendsen Pcoupltype = semiisotropic tau\_p = 5.0 compressibility = 3e-4 3e-4 ref\_p = 1.0 1.0 ; GENERATE VELOCITIES FOR STARTUP RUN: gen\_vel = yes gen\_temp = 323.15 gen\_seed = 6015460206 ;soft-core-minimization so that single precision GROMACS works here ; Free energy parameters free-energy = yes init-lambda = 0.01 sc-alpha = 4 sc-power = 2 sc-coul = yes nstdhdl = 0 couple-moltype = system ; we are changing both the vdw and the charge. In the initial state, both are on couple-lambda0 = vdw-q ; in the final state, both are off. couple-lambda1 = none couple-intramol = yes refcoord\_scaling = all ---
**Author:** ![kyrstyno](https://avatars.discourse-cdn.com/v4/letter/k/d07c76/32.png) [@kyrstyno](https://gromacs.bioexcel.eu/u/kyrstyno)\ **Post date:** [August 12, 2022, 2:32am UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/4 "2022-08-12T02:32:48Z")
Also because the system is around 30 by 30 by 20 nm, does it still make sense to continue increase the rvdw and rcoulomb at all beyond 10 nm? I am not sure exactly what the limit of the cutoffs should be. ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [August 12, 2022, 2:43am UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/5 "2022-08-12T02:43:53Z")
> [@kyrstyno](#): > > In that case do you recommend turning off the free energy? I am not performing perturbations on the system yet. Well, you are by virtue of setting a non-zero value of λ, which I presume is being used to soften interactions and help resolve clashes. This is a strategy I would generally only recommend for problematic systems, but in your case it seems to be causing problems. I would try a normal minimization and equilibration protocol before trying to do anything unorthodox. > [@kyrstyno](#): > > Also because the system is around 30 by 30 by 20 nm, does it still make sense to continue increase the rvdw and rcoulomb at all beyond 10 nm? I am not sure exactly what the limit of the cutoffs should be. You should not mess with cutoffs; they are (generally considered) part of the force field. There are other options that govern DD cell size and such that could be tweaked in special cases, but again I wouldn’t be messing with free energy options here. ---
**Author:** ![kyrstyno](https://avatars.discourse-cdn.com/v4/letter/k/d07c76/32.png) [@kyrstyno](https://gromacs.bioexcel.eu/u/kyrstyno)\ **Post date:** [August 12, 2022, 3:43am UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/6 "2022-08-12T03:43:35Z")
Thank you. I was using the default CHARMM-GUI file, and then the only things I initially tried to vary were rcoulomb and rvdw since it said in the README file “step6.0 - soft-core minimization. If you encountered ‘There are 1 perturbed non-bonded pair interaction …’ error message, please modify rvdw and rcoulomb values from 1.1 to 2.0 in the step6.0\_minimization.mdp file.” I have modified the mdp file to remove the free energy section, but now I am running into a different error regarding infinite forces. Output: Steepest Descents: Tolerance (Fmax) = 1.00000e+01 Number of steps = 5000 Energy minimization has stopped because the force on at least one atom is not finite. This usually means atoms are overlapping. Modify the input coordinates to remove atom overlap or use soft-core potentials with the free energy code to avoid infinite forces. You could also be lucky that switching to double precision is sufficient to obtain finite forces. writing lowest energy coordinates. Steepest Descents converged to machine precision in 15 steps, but did not reach the requested Fmax \< 10. Potential Energy = 8.7619801e+23 Maximum force = inf on atom 15123 Norm of force = inf mdp file: define = -DFLEXIBLE integrator = steep tinit = 0.0 nsteps = 5000 nstlog = 100 nstenergy = 100 nstxout-compressed = 1000 compressed-x-precision = 100 cutoff-scheme = Verlet nstlist = 1 ns\_type = grid pbc = xyz verlet-buffer-tolerance = 0.005 epsilon\_r = 15 coulombtype = reaction-field rcoulomb = 2 vdw\_type = cutoff vdw-modifier = Potential-shift-verlet rvdw = 2 tcoupl = v-rescale tc-grps = protein membrane solute tau\_t = 1.0 1.0 1.0 ref\_t = 323.15 323.15 323.15 ; Pressure coupling: Pcoupl = berendsen Pcoupltype = semiisotropic tau\_p = 5.0 compressibility = 3e-4 3e-4 ref\_p = 1.0 1.0 ; GENERATE VELOCITIES FOR STARTUP RUN: gen\_vel = yes gen\_temp = 323.15 gen\_seed = 6015460206 refcoord\_scaling = all ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [August 12, 2022, 7:41pm UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/7 "2022-08-12T19:41:46Z")
It seems your system has some very nasty contacts. Try minimizing with the free energy options enabled, then minimize those coordinates again without them to see if you need to continue using the softened potential for any future steps. ---
**Author:** ![kyrstyno](https://avatars.discourse-cdn.com/v4/letter/k/d07c76/32.png) [@kyrstyno](https://gromacs.bioexcel.eu/u/kyrstyno)\ **Post date:** [August 12, 2022, 7:44pm UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/8 "2022-08-12T19:44:38Z")
Thank you! I will try that. This particular ion channel is known to induce a membrane indentation/dome around it at 1 bar pressure, but since I can only initialize the lipids in a planar configuration, I am expecting that through minimization the lipids will start to fill in around the blades of the ion channel to form the dome. However, I am starting to think that I should instead take the ion channel pdb file and relax the protein structure in a vacuum until it is in its planar configuration (by adding negative pressure in the lateral dimension to stretch it out) before building the membrane around it. If I energy minimize and equilibrate a protein on its own in a vacuum, is is possible to add negative pressure in the lateral dimension without completely deforming the channel? ![my_Piezo1_membrane](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/0/0a8875674dfe05ac51da2b94907078344bf4f37c.jpeg) ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [August 13, 2022, 1:53am UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/9 "2022-08-13T01:53:15Z")
You can’t apply pressure when doing something in vacuum, the box will collapse down onto the protein and you’ll start simulating a crystal. ---
**Author:** ![Amy](https://avatars.discourse-cdn.com/v4/letter/a/4da419/32.png) [@Amy](https://gromacs.bioexcel.eu/u/Amy)\ **Post date:** [March 19, 2024, 4:37am UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/10 "2024-03-19T04:37:50Z")
Hi @kyrstyno and @jalemkul, I’m working on Piezo1 ion channel embedded in membrane system and encountered the same problem as @kyrstyno. “Fatal error: There are 1 perturbed non-bonded pair interactions beyond the pair-list cutoff of 2.1 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.” My .mdp file is similar to @kyrstyno as well because I also used CHARMM-GUI for building the system. As suggested, I tried couple-intramol=yes, increase rlist and nstlist, but still get the same error. When I tried minimizing without free energy options, or minimizing with the free energy options enabled then minimize those coordinates again without them, I also got the same error as @kyrstyno regarding infinite forces. I’m not sure what’s happening here and what should I do to get this fix and get my minimization running. I really appreciate any advice/suggestions. @kyrstyno: Have you by any chance fixed this error? ---
**Author:** ![Amy](https://avatars.discourse-cdn.com/v4/letter/a/4da419/32.png) [@Amy](https://gromacs.bioexcel.eu/u/Amy)\ **Post date:** [March 20, 2024, 2:07pm UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/11 "2024-03-20T14:07:25Z")
@jalemkul Would you have any further insights/suggestions on this problem? ---
**Author:** ![hess](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/hess/32/416_2.png) [@hess](https://gromacs.bioexcel.eu/u/hess)\ **Post date:** [March 20, 2024, 5:30pm UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/12 "2024-03-20T17:30:16Z")
You seem to be discussing the same issue in two threads now which is confusing and can lead to more work for the people answering. ---
**Author:** ![Amy](https://avatars.discourse-cdn.com/v4/letter/a/4da419/32.png) [@Amy](https://gromacs.bioexcel.eu/u/Amy)\ **Post date:** [March 20, 2024, 6:52pm UTC](https://gromacs.bioexcel.eu/t/perturbed-non-bonded-pair-interaction-beyond-the-pair-list-cutoff/4596/13 "2024-03-20T18:52:28Z")
I’m sorry for any confusion this may cause. I came across this thread from @kyrstyno with the same system with the exact same errors, so I just wanted to ask if he/she has figured out the solution, which would be applicable for my system as well.