partly interacting and blocking or slowing solvent access to this space, this motion was slow to sample. For multiple λ windows, the number of water molecules in the binding site differed among the independent replicates, and 40 ns sampling time/λ window was not always sufficient to reach equilibrium. Jumps in dH/dλ curves correlated with water molecules entering the buried space (Figure ). This suggests that sampling the correct number of waters in the binding site has an impact on the ∆G estimate. For the fully decoupled ligand, the timescales of the water molecules filling the empty binding site were relatively fast (roughly a few hundred ps).