Changes in water placement and occupancy on binding can lead to sampling problems in HSP90. In the unbound state, the solvent completely fills the binding site. In the presence of the ligand, three water molecules are deeply buried in a space between the ligand and the protein (Figure ). In our MD simulations where these water molecules were not included in the starting structure, we found they do not enter the space even after 40 ns (Sec. 5.5.4). We ran calculations both with and without these bound water molecules to examine their con-tribution to the binding free energy. In the EQ approach, we studied whether the use of HREX alleviates the sampling of this slow motion.