# Free energy calculations error **URL:** \ **Category:** pmx\ **Created:** [April 14, 2022, 4:37pm UTC](https://ask.bioexcel.eu/t/free-energy-calculations-error/3672 "2022-04-14T16:37:24Z")\ **Posts on this page:** 2\ **Page:** 1
**Author:** ![cyber222](https://avatars.discourse-cdn.com/v4/letter/c/3be4f8/32.png) [@cyber222](https://ask.bioexcel.eu/u/cyber222)\ **Post date:** [April 14, 2022, 4:37pm UTC](https://ask.bioexcel.eu/t/free-energy-calculations-error/3672/1 "2022-04-14T16:37:24Z")
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. 1. Downloaded PDB structure from RCSB 2. Splitted it into several chains(because I dont know how to rebuild missing residues in in multimeric protein) 3. Rebuilt missing residues in every chain using Modeller 4. Combined all chains together 5. Created hybrid topology using PMX 6. 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 ---
**Author:** ![vgapsys](https://avatars.discourse-cdn.com/v4/letter/v/7993a0/32.png) [@vgapsys](https://ask.bioexcel.eu/u/vgapsys)\ **Post date:** [June 14, 2022, 3:17pm UTC](https://ask.bioexcel.eu/t/free-energy-calculations-error/3672/2 "2022-06-14T15:17:42Z")
Warning 1: what is exactly in the line 2692 of hybrid\_posre\_Protein\_chain\_A.itp? Warning 2: this is because you are using h-bond constraints in combination with the ‘master’ branch of pmx. For that you need all-bond constraints. If you want to simulate with h-bond constraints only, you will need to switch to the ‘develop’ branch of pmx. Does the energy minimization finish properly? What is the maximum force you get? Is the geometry of the system retained? In particular, does the hybrid residue with the dummy atoms appear correct? Vytas