The overall binding free energy of the HSP90 ligand, which was obtained through the thermodynamic cycle, varied between -11.0 and -11.8 kcal/mol among different protocols, as shown in SI Table . The binding free energy of the protocols, where three buried water molecules were not present in the starting structure (Sec. 5.5.4), differs from the other protocols by ∼ 2 kcal/mol (-8.6 to -9.9 kcal/mol). These protocols agreed best with the experimental binding free energy of 9.34 +/-0.07 kcal/mol, although inadequate sampling was most severe. This is possible since agreement with experimental values is not only a function of adequate sampling, but also of the force field and other factors. Therefore, we focus on whether we have obtained converged values rather than on agreement with experimental values. 5.5.1. Slow water sampling in the solvent exposed binding site of HSP90. The first issue we identified was slow water sampling in this solvent exposed binding site. In the unbound state, water molecules completely fill the binding site. Upon binding the ligand displaces the solvent molecules except three buried water molecules between the ligand and the protein (Figure ).