As noted above, the ABFE calculations predict that some decoy compounds have quite favorable binding free energies. Some of these DUD-E decoys may truly be active , especially as our docking calculations should have enriched the fraction of true actives among the high-ranking decoys studied here. However, we believe the apparent overestimation of many decoy binding affinities reflects imperfections in the ABFE calculations. Two possible explanations come to mind. The first is that errors in the force field may cause these affinities to be overestimated. If so, this would most likely result from an imbalance in nonbonded interactions, i.e., in the electrostatic and Lennard-Jones terms of the protein, the ligand, and/or the aqueous solvent. Interestingly, two lines of evidence suggest that standard water models lead to overestimation of effective intramolecular protein attractions and host-guest attractions and have motivated adjustments to the TIP4P and TIP3P models, respectively. It is possible that same imbalance leads here overestimation and that these modified water models would reduce or abolish the apparent overestimations observed here. Also, given the diversity of ligand chemical structures, bespoke ligand force field parameters optimized separately for each ligand may model the ligand conformational landscape more accurately . The second possible explanation is that our ABFE calculations may not adequately capture the fall in free energy associated with relaxation of the ligand-bound protein to its unbound conformational ensemble following decoupling of the bound ligand . Underestimating this fall would cause the free energy of dissociation to appear less favorable than it ought and therefore make the binding free energy overly favorable.