The presence of three buried water molecules in the binding site in the starting structure was crucial for both the EQ and NEQ approach. Indeed, not including these crystallographic waters in the starting structure had a great impact on the computed free energy difference as shown in Figure . Only when the ligand was already mostly decoupled, water molecules entered the binding site (SI). Using HREX in the EQ approach did not enhance the sampling sufficiently (Figure , red line), and other enhanced sampling methods, like GC/MC or MC/MD, are likely necessary here unless far longer simulations are used, which would raise computational costs far beyond what is currently standard for these calculations. Interestingly, inserting the ligand with NEQ transitions "found" these three water molecules in exactly the same positions as in the crystal structure.