We prevented the solvent from flooding the binding site, once the trapped water was decoupled, by applying a solvent repulsion term before the trapped water decoupling (edge C, Figure ). The solvent repulsion term is subsequently removed in our thermodynamic cycle, only after the trapped water is decoupled (edge G, Figure ). Upon removal of the solvent repulsion term, the solvent could flood the binding site, which could have subsequently resulted in sampling issues in the simulation of an unrestrained state with a cavity in the binding site (stage 7, Figure ). For one of the three replicates of the HIV-1 Protease system 1EC0, we did observe solvent flooding of the cavity in the simulation of the unrestrained state. However, our calculations resulted in a dry cavity in the unrestrained state, when we started the simulation of the unrestrained state from a structure that had time to adapt to the cavity in the binding site, i.e., the equilibrated structure of the system with a non-interacting water in the binding site, and the solvent repulsion in place (stage 6, Figure ).