For non-equilibrium transitions, simulation settings remain the same; the simulations are initiated from equidistant conformations chosen from production runs of the equilibrium simulations at the two end-states. The number of conformations chosen in each direction (in other words, the number of forward and reverse transitions) and their lengths are NEQ specific-parameters that cannot be chosen arbitrarily as done in all previous ABFE studies using the NEQ approach. The only exception is the study by Gapsys et al. where the transition length was varied up to 20 ns to show that 500 ps was optimal for a small set of 7 ligands. We have performed a systematic investigation of the effect of these parameters on ABFE calculations using a large dataset and recommended optimal values for each as discussed in the Results section. For this study, we have generally used our recommended "standard" protocol (see the Discussion section for detail). This requires performing 500/250 non-equilibrium transitions in each direction with protein/water environment starting from equidistant equilibrium conformations (50 per replica chosen at an interval of 200 ps taking an ensemble size of 10/5) per ABFE prediction.