Although the present ABFE calculations successfully enhance the enrichment of known actives from decoys, we find essentially no correlation between our computed ABFEs and the available affinity data for the actives, as provided in the DUD-E dataset used here (Figure ). This contrasts with prior ABFE studies which, using similar force fields, have obtained significant correlations between calculation and experiment . We believe this difference results primarily from differences in the accuracy of the ligand poses used to initiate the ABFE calculations. Here, we have deliberately replicated a virtual screening setting where there is minimal prior information about the poses of the chemically diverse ligands to be screened. Accordingly, our initial poses were generated by unsupervised docking into a single protein crystal structure for each protein target. Although we used a reasonable docking protocol, docking is still far from exact, and the imperfections and inconsistencies in our starting poses undoubtedly contributed to the errors in our ABFE results. In contrast, prior tests of ABFE methods appear to have utilized relevant structural information to obtain relatively refined poses than is available in the setting of a real world virtual screening campaign. In particular, some studies have applied their ABFE methods to congeneric series of ligands, where a consistent set of starting poses could be generated by overlaying the compounds' common chemical scaffold on that observed in a co-crystal structure for one of the compounds in the series ; and other studies considered chemically diverse ligands but started each ABFE calculation from the available co-crystal structures of the respective ligand .