In equilibrium FEC, inadequate water sampling was mainly observed in the intermediate λ windows. As the interactions of the ligand were decoupled, more water molecules entered the space between the ligand and the protein, joining the three buried water molecules (Figure ). However, since the ligand was still Figure . In the HSP90 system, water molecules got trapped in the binding site when the ligand appeared quickly during NEQ transitions as well as in intermediate λ windows in the EQ approach. HSP90 is shown in green, ligand in yellow sticks and water molecules are depicted as spheres.