We would also like to investigate what effect the binding site has on acceptance rates of these NCMC moves. The T4 lysozyme binding cavity we tested on is fairly rigid; however, it could be more difficult to achieve high acceptance rates with this approach in systems with solvent-exposed or flexible binding sites, due to protein or water rearrangements as the ligand is turned off. Preliminary tests with BLUES rotational moves of toluene in solution (without protein) show reasonable acceptance rates (SI Figure ), suggesting that water rearrangements, at least, would not cause major issues with this methodology. Another potential concern is that the ligand could more easily leave the binding site when its interactions are turned off. We have not observed such an effect here, probably in part because our moves are purely rotational and the time spent noninteracting is minimal. However, if this were to occur, modest restraints could perhaps be used to prevent this.