Therefore, to calculate the correct restraint potential, we post-processed the trajectory of stage 1, such that the water from the bulk solvent that is occupying the trapped water's binding site, would be considered for the calculation of the restraint potential in the MBAR calculation. We did this by re-mapping/reindexing the trajectory waters to ensure the nearest water is always the one considered for restraint calculation. Without this re-indexing, we would encounter challenges in systems where the trapped water is (partially) exposed to the bulk solvent and may periodically exchange with a water molecule from the bulk solvent in stage 1 (in later stages, the restraint prevents the exchange). Because water molecules are indistinguishable, such an exchange should not influence the restraint potential. When calculating the Hamiltonian of later stages with the trajectory of stage 1, we hence want to apply the harmonic restraint to the water molecule currently occupying the binding site in stage 1, and not track a molecule that has left the binding site and diffused into the solvent. Failure to treat the water molecules as indistinguishable in stage 1 could lead to very large free energies and poor overlap between stage 1 and its neighboring stage (stage 2). Thus, we here implemented proper treatment of indistinguishable water molecules through post-processing.