However, it cannot be arbitrarily short as for reliable estimates the NEQ approach requires the work values obtained from such transitions in the forward and reverse directions to be overlapping. If the transition length is too small, the work dissipated would be large and hence end up being very different in the two directions leading to widely varying values and compromising the reliability of predicted free energies. On the other hand, longer transitions make the distributions narrower while bringing them closer to each other and also decrease the overlap . Futher, they add substantially to the cost of each ABFE calculation. Therefore, we need to identify a "sweet spot" for the transition length. We reported that the minimum transition length in case of RBFE calculations should be 250 ps . In the literature, transition lengths of around 500 ps have been used for ABFEs without any justification . Gapsys et al. attempted to investigate this aspect to arrive at 500 ps but their dataset was extremely small (just 7 ligand-protein complexes with a rigid protein) and hence their recommendation lacks statistical robustness. With flexible protein-ligand complexes, it has been reported that even 10 ns transition length can be insufficient for "tricky" cases . In this study we recommend an optimal value for this parameter based on rigorous and systematic analysis. It is clear that there is no one-size-fit-all solution; rather this parameter needs to be selected flexibly. We used a subset of 34 ligands binding to P38 to investigate this parameter. We varied the transition lengths for this subset from 100 ps to 2 ns in steps. Table compares the effect of varying the transition length on ABFE predictions. MUE (compared with experiment) corresponding to ABFE predictions using different transition lengths is provided. Further, the fraction of complexes (in % terms) with error bars less than or equal to 0.5 kcal/mol and greater than 1 kcal/mol are included for each case along with ranges of uncertainties. Figure displays the corresponding correlation plots for each case including the fraction of complexes with unsigned error less than 1 kcal/mol in the inset. It should be noted that to obtain this data all other parameters were kept constant (ensemble size 10 (5 for ligand in water), end-point replica length 10 ns, 50 transitions per replica). It is clear that with increasing transition lengths, both accuracy and precision of predicted free energies improves consistently. Figure displays the distribution of ∆G com using transition lengths 1 ns, 2 ns and 4 ns for 9 complexes of the BRD4-2 system. It is evident that, on increasing the transition length from 1 ns to 2 ns, ∆G com becomes more negative, but a further increase to 4 ns does not change ∆G com anymore.