Both water sampling and the rearrangement of the Asn36 side chain were improved by HREX mixing of replicates. Sampling of the correct binding mode, however, seemed to be more challenging, since unfavorable (flipped) binding modes were passed between different alchemical states through replica exchange swaps (SI). This was possible because some alternate binding modes were favorable enough to be swapped to alchemical states retaining significant interactions and became temporarily trapped, but not so favorable that they ought to be populated substantially at equilibrium. We reran the analysis after discarding all data that included the flipped binding mode. This changed the estimate of the free energy change by 0.4 kcal/mol and shows the impact of the binding mode flip (Figure ).