After decoupling the trapped water from the system and removing the binding site restraints, the binding site may change its conformation in stage 6. This can result in the stages 6 and 5 being distant in phase space, and requires a free energy calculation routine that can adequately sample the conformational change. We therefore introduced 7 intermediate λ states between stages 5 (fully restrained) and 6 (fully unrestrained), and reduced the position restraint on the binding site across each subsequent λ state, using force constants amounting to 64%, 32%, 16%, 8%, 4%, 2%, and 1% of the force constant of the full position restraints in stage 5 (see Section S1.1 and Table in the Supporting Document for details). Throughout the simula-tions of the intermediate λ states, the restraint on the trapped water and the solvent repulsion were kept in place. 2.4. The thermodynamic cycle to calculate the RBFE between ligands involving displacement of a trapped water . The RBFE between two ligands is the difference between the free energies to transform one ligand into the other in the complex (complex leg) and in the solvent (solvent leg) (Figure ). For the complex leg of the RBFE calculation, we revised the ABFE thermodynamic cycle for the trapped water into an RBFE thermodynamic cycle that handles displacement of a trapped water upon modification of a ligand (Figure ). In particular, we imagine this RBFE thermodynamic cycle will be used in cases where prior analysis has determined that modification of a particular initial ligand into another ligand will displace a relatively ordered (and especially a slow) water which requires separate handling in the free energy calculation.