I am carrying out an alchemical free energy calculations for amino acid mutation. When I performed non-equilibrium transition step, I had an issue. When I performed 50ps transition, the transition step worked perfectly without any error, however, when I increased to 100ps transition, gromacs notified me that

" Step 26048: The total potential energy is -nan, which is not finite. The LJ and electrostatic contributions to the energy are 653461 and -nan, respectively. A non-finite potential energy can be caused by overlapping interactions in bonded interactions or very large or Nan coordinate values. Usually this is caused by a badly- or non-equilibrated initial configuration, incorrect interactions or parameters in the topology."

Before the non-equilibrium transition step, I carried out 2 steps of energy minimization (1 step with steepest descent algorithm and another one with lbfgs. It is because when performing em with only steepest descent, my system can not converge the force to the requested precision Fmax < 100), 500 ps of nvt and 5ns of npt.

Is there anyone faced this issue before? Thank you so much for your attention.

p/s: I used double-system/single box approach for this simulation because my mutation induces net charge change.