The direct method involves a much larger amount of sampling as compared to ESMACS-s that involves performing short MD simulations of only a few nanoseconds duration. It is, however, notable that ESMACS-s is still able to obtain almost identical ranking of ligand-protein complexes with such little sampling which makes it a much more efficient method when accuracy is not necessary. Nevertheless, it is well known that ESMACS-s results depend heavily on the initial binding pose/structure of the ligand-protein complex being studied due to the short duration of simulations; this can be a drawback in some cases where the initial structure is not known correctly. In such cases, ESMACS-s is not so useful as resultant ∆G values may vary substantially (see specific details in the supporting information). On the other hand, due to substantially more sampling, the direct free energy method is expected to overcome this drawback. In this study, we have performed ESMACS calculations using all "bound" conformations (as defined so during ∆G direct calculation) extracted from all "long" trajectories such that the ensemble averaging is performed across all replicas that sample a given binding site (denoted as "ESMACS-l"). Free energies so obtained are expected to be free from the dependence on starting structures and better correlated with ∆G direct . This is evident in Figure where ∆G ESM ACS-l are consistently more negative than ∆G ESM ACS-s and have a higher correlation coefficient of 0.95. This is because all the different binding poses sampled during the long duration of simulations have been taken into account with appropriate weights. The key conclusion is that it is sufficient to apply ESMACS-s for ranking ligands based on their binding affinities when the starting structure is confidently known; otherwise extensive sampling becomes necessary.