The B3 simulations were also rerun with protein anchor atoms on a different residue (Ala38A) -see B3-P. There was very good agreement between runs 2 -5 for the bound vanish stage, but ∆G o Bound was around 3 kcal mol -1 more negative for run 1 due to comparatively low number of waters in the binding site over just 3 λ windows (see Section S9). This highlights the importance of correctly sampling rehydration of the binding site upon decoupling. It has been demonstrated that hybrid sampling approaches combining molecular dynamics with Monte Carlo water moves, in both the µV T and N P T ensembles, can improve the performance of relative binding free energy calculations. In this system, it is possible that these approaches may perform poorly as a result of low acceptance probabilities, because water has to strain the partially decoupled ligand to enter the binding site around λ = 0.4, where proper sampling of rehydration is most critical. Non-equilibirum candidate Monte Carlo may overcome this by allowing for relaxation of the ligand position. The strong dependence of the free energy on the correct sampling of binding-site water may make this a good system for testing methods for enhancing sampling of rehydration. Regardless, the