Hi everyone! I am facing error in minimization step during simulation of a protein. I have used amber99sb-ILDN forcefield. for this forcefield, Br ion parameters were available in ffnonbonded.itp and atomtypes.itp but it was not there in ions.itp. so, i added Br in ions.itp and added Li and Br with conc. 0.15 as counter ions to neautalise the system. but in the minimization step I am facing error.

Command line: gmx_mpi mdrun -v -deffnm em

Reading file em.tpr, VERSION 2021.4 (single precision) Using 1 MPI process Using 32 OpenMP threads

Steepest Descents: Tolerance (Fmax) = 1.00000e+03 Number of steps = 50000 Step= 0, Dmax= 1.0e-05 nm, Epot= -1.88811e+05 Fmax= 1.40288e+05, atom= 131 Step= 1, Dmax= 1.0e-05 nm, Epot= -1.88851e+05 Fmax= 1.40111e+05, atom= 131 Step= 2, Dmax= 1.2e-05 nm, Epot= -1.88899e+05 Fmax= 1.39901e+05, atom= 131 Step= 3, Dmax= 1.4e-05 nm, Epot= -1.88957e+05 Fmax= 1.39651e+05, atom= 131 Step= 4, Dmax= 1.7e-05 nm, Epot= -1.89026e+05 Fmax= 1.39348e+05, atom= 131 Step= 5, Dmax= 2.1e-05 nm, Epot= -1.89109e+05 Fmax= 1.38987e+05, atom= 131 Step= 6, Dmax= 2.5e-05 nm, Epot= -1.89208e+05 Fmax= 1.38555e+05, atom= 131 Step= 7, Dmax= 3.0e-05 nm, Epot= -1.89328e+05 Fmax= 1.38038e+05, atom= 131 Step= 8, Dmax= 3.6e-05 nm, Epot= -1.89471e+05 Fmax= 1.37422e+05, atom= 131 Step= 9, Dmax= 4.3e-05 nm, Epot= -1.89642e+05 Fmax= 1.36686e+05, atom= 131 Step= 10, Dmax= 5.2e-05 nm, Epot= -1.89846e+05 Fmax= 1.35805e+05, atom= 131 Step= 11, Dmax= 6.2e-05 nm, Epot= -1.90091e+05 Fmax= 1.34755e+05, atom= 131 Step= 12, Dmax= 7.4e-05 nm, Epot= -1.90383e+05 Fmax= 1.33506e+05, atom= 131 Step= 13, Dmax= 8.9e-05 nm, Epot= -1.90733e+05 Fmax= 1.32019e+05, atom= 131 Step= 14, Dmax= 1.1e-04 nm, Epot= -1.91149e+05 Fmax= 1.30254e+05, atom= 131 Step= 15, Dmax= 1.3e-04 nm, Epot= -1.91646e+05 Fmax= 1.28159e+05, atom= 131 Step= 16, Dmax= 1.5e-04 nm, Epot= -1.92237e+05 Fmax= 1.25682e+05, atom= 131 Step= 17, Dmax= 1.8e-04 nm, Epot= -1.92939e+05 Fmax= 1.22763e+05, atom= 131 Step= 18, Dmax= 2.2e-04 nm, Epot= -1.93771e+05 Fmax= 1.19338e+05, atom= 131 Step= 19, Dmax= 2.7e-04 nm, Epot= -1.94755e+05 Fmax= 1.15334e+05, atom= 131 Step= 20, Dmax= 3.2e-04 nm, Epot= -1.95916e+05 Fmax= 1.10673e+05, atom= 131 Step= 21, Dmax= 3.8e-04 nm, Epot= -1.97282e+05 Fmax= 1.05283e+05, atom= 131 Step= 22, Dmax= 4.6e-04 nm, Epot= -1.98883e+05 Fmax= 9.90926e+04, atom= 131 Step= 23, Dmax= 5.5e-04 nm, Epot= -2.00754e+05 Fmax= 9.20480e+04, atom= 131 Step= 24, Dmax= 6.6e-04 nm, Epot= -2.02933e+05 Fmax= 8.41092e+04, atom= 131 Step= 25, Dmax= 7.9e-04 nm, Epot= -2.05462e+05 Fmax= 7.64873e+04, atom= 7598 Step= 26, Dmax= 9.5e-04 nm, Epot= -2.08343e+05 Fmax= 6.83805e+04, atom= 7598 Step= 27, Dmax= 1.1e-03 nm, Epot= -2.11600e+05 Fmax= 5.98322e+04, atom= 7598 Step= 28, Dmax= 1.4e-03 nm, Epot= -2.15259e+05 Fmax= 5.10455e+04, atom= 7598 Step= 29, Dmax= 1.6e-03 nm, Epot= -2.19345e+05 Fmax= 4.22813e+04, atom= 7598 Step= 30, Dmax= 2.0e-03 nm, Epot= -2.23886e+05 Fmax= 3.40412e+04, atom= 492 Step= 31, Dmax= 2.4e-03 nm, Epot= -2.28891e+05 Fmax= 2.83132e+04, atom= 1595 Step= 32, Dmax= 2.8e-03 nm, Epot= -2.34057e+05 Fmax= 2.40559e+04, atom= 1595 Step= 33, Dmax= 3.4e-03 nm, Epot= -2.39206e+05 Fmax= 1.98149e+04, atom= 1595 Step= 34, Dmax= 4.1e-03 nm, Epot= -2.44464e+05 Fmax= 1.57188e+04, atom= 1595 Step= 35, Dmax= 4.9e-03 nm, Epot= -2.49959e+05 Fmax= 1.20305e+04, atom= 8381 Step= 36, Dmax= 5.9e-03 nm, Epot= -2.55801e+05 Fmax= 9.30679e+03, atom= 8381 Step= 37, Dmax= 7.1e-03 nm, Epot= -2.61892e+05 Fmax= 7.01439e+03, atom= 8381 Step= 38, Dmax= 8.5e-03 nm, Epot= -2.68506e+05 Fmax= 5.09389e+03, atom= 8381 Step= 39, Dmax= 1.0e-02 nm, Epot= -2.76081e+05 Fmax= 3.52033e+03, atom= 2231 Step= 40, Dmax= 1.2e-02 nm, Epot= -2.85188e+05 Fmax= 2.49421e+03, atom= 2231 Step= 41, Dmax= 1.5e-02 nm, Epot= -2.95630e+05 Fmax= 4.17521e+03, atom= 596 Step= 42, Dmax= 1.8e-02 nm, Epot= -3.00591e+05 Fmax= 2.12663e+04, atom= 596 Step= 43, Dmax= 2.1e-02 nm, Epot= -3.01982e+05 Fmax= 1.11674e+04, atom= 596 Step= 44, Dmax= 2.5e-02 nm, Epot= -3.03980e+05 Fmax= 2.71631e+04, atom= 596 Step= 45, Dmax= 3.0e-02 nm, Epot= -3.05338e+05 Fmax= 1.95931e+04, atom= 596 Step= 46, Dmax= 3.7e-02 nm, Epot= -3.06607e+05 Fmax= 3.56677e+04, atom= 596 Step= 47, Dmax= 4.4e-02 nm, Epot= -3.07972e+05 Fmax= 3.10439e+04, atom= 596 Step= 48, Dmax= 5.3e-02 nm, Epot= -3.08790e+05 Fmax= 4.78081e+04, atom= 596 Step= 49, Dmax= 6.3e-02 nm, Epot= -3.10296e+05 Fmax= 4.64267e+04, atom= 596 Step= 50, Dmax= 7.6e-02 nm, Epot= -3.19027e+05 Fmax= 1.83250e+06, atom= 23043 Step= 51, Dmax= 9.1e-02 nm, Epot= -3.10219e+05 Fmax= 5.97203e+04, atom= 596 Step= 52, Dmax= 4.6e-02 nm, Epot= -3.10312e+05 Fmax= 6.19996e+04, atom= 596 Step= 53, Dmax= 2.3e-02 nm, Epot= -3.11147e+05 Fmax= 6.39242e+04, atom= 23043 Step= 54, Dmax= 1.1e-02 nm, Epot= -3.13970e+05 Fmax= 4.25138e+05, atom= 23043 Step= 55, Dmax= 5.7e-03 nm, Epot= -3.27429e+05 Fmax= 5.94123e+06, atom= 23043 Step= 56, Dmax= 6.8e-03 nm, Epot= -3.15113e+05 Fmax= 6.95023e+05, atom= 23043 Step= 57, Dmax= 3.4e-03 nm, Epot= -3.27575e+05 Fmax= 6.06913e+06, atom= 23043 Step= 58, Dmax= 4.1e-03 nm, Epot= -3.20865e+05 Fmax= 2.48044e+06, atom= 23043 Step= 59, Dmax= 2.0e-03 nm, Epot= -3.72426e+05 Fmax= 6.97644e+07, atom= 23043 Step= 60, Dmax= 2.5e-03 nm, Epot= -3.23472e+05 Fmax= 3.69377e+06, atom= 23043 Step= 61, Dmax= 1.2e-03 nm, Epot= -3.46829e+05 Fmax= 2.49015e+07, atom= 23043 Step= 62, Dmax= 6.1e-04 nm, Epot= -4.02587e+05 Fmax= 7.60497e+07, atom= 23043 Step= 63, Dmax= 7.4e-04 nm, Epot= -3.58745e+05 Fmax= 4.29664e+07, atom= 23043 Step= 64, Dmax= 3.7e-04 nm, Epot= -4.02588e+05 Fmax= 1.04434e+08, atom= 23043 Step= 65, Dmax= 4.4e-04 nm, Epot= -4.02587e+05 Fmax= 1.12412e+08, atom= 23043 Step= 66, Dmax= 2.2e-04 nm, Epot= -4.02587e+05 Fmax= 4.14473e+06, atom= 23043 Step= 67, Dmax= 1.1e-04 nm, Epot= -4.02588e+05 Fmax= 5.02694e+07, atom= 23043 Step= 68, Dmax= 5.5e-05 nm, Epot= -4.02588e+05 Fmax= 7.73021e+07, atom= 23043 Step= 69, Dmax= 2.8e-05 nm, Epot= -4.02588e+05 Fmax= 9.08984e+07, atom= 23043 Step= 70, Dmax= 1.4e-05 nm, Epot= -4.02588e+05 Fmax= 9.77319e+07, atom= 23043 Step= 71, Dmax= 6.9e-06 nm, Epot= -4.02588e+05 Fmax= 1.01115e+08, atom= 23043 Step= 72, Dmax= 3.5e-06 nm, Epot= -4.02588e+05 Fmax= 1.02661e+08, atom= 23043 Step= 73, Dmax= 1.7e-06 nm, Epot= -4.02588e+05 Fmax= 1.03568e+08, atom= 23043 Energy minimization has stopped, but the forces have not converged to the requested precision Fmax < 1000 (which may not be possible for your system). It stopped because the algorithm tried to make a new step whose size was too small, or there was no change in the energy since last step. Either way, we regard the minimization as converged to within the available machine precision, given your starting configuration and EM parameters.

Double precision normally gives you higher accuracy, but this is often not needed for preparing to run molecular dynamics. You might need to increase your constraint accuracy, or turn off constraints altogether (set constraints = none in mdp file)

writing lowest energy coordinates.

Steepest Descents converged to machine precision in 74 steps, but did not reach the requested Fmax < 1000. Potential Energy = -4.0258788e+05 Maximum force = 1.0443410e+08 on atom 23043 Norm of force = 9.7191676e+05

please find the em.mdp file used:

; minim.mdp - used as input into grompp to generate em.tpr ; Parameters describing what to do, when to stop and what to save integrator = steep ; Algorithm (steep = steepest descent minimization) emtol = 1000.0 ; Stop minimization when the maximum force < 1000.0 kJ/mol/nm emstep = 0.01 ; Minimization step size nsteps = 50000 ; Maximum number of (minimization) steps to perform

; Parameters describing how to find the neighbors of each atom and how to calculate the interactions nstlist = 20 ; Frequency to update the neighbor list and long range forces cutoff-scheme = Verlet ; Buffered neighbor searching ns_type = grid ; Method to determine neighbor list (simple, grid) coulombtype = PME ; Treatment of long range electrostatic interactions rcoulomb = 1.0 ; Short-range electrostatic cut-off rvdw = 1.0 ; Short-range Van der Waals cut-off pbc = xyz ; Periodic Boundary Conditions in all 3 dimensions

please help me to rectify this error. I am new to gromacs. I have read many discussion related to this error.but i was unsuccessful. Requesting you for step by step resolution.