As our separation of states-based method requires prior knowledge of numbers and locations of the trapped water in contexts of each of the ligands, we are working on a separate study on how to best predict the water positions and occupancies in protein-ligand complexes. Enhanced sampling simulation methods, such as GCMC and its variants, can also be used to determine the locations of trapped waters, before performing the free energy calculation with our protocol. For example, a recent study combining GCMC with MD simulation for sampling the trapped waters in ligand RBFE calculations showed that most of the advantage offered by GCMC comes from better water sampling during the equilibration phase of simulations. While that study also tested GCMC during free energy calculations, most of the benefit came simply from better equilibration. Thus, better water equilibration (to access the numbers and locations of trapped water) -such as that offered by GCMC -will likely be sufficient in many cases, and may even be more efficient since it will not require mixing GCMC sampling in to binding free energy calculations. In our case, then, a GCMC equilibration of the initial structure, prior to water decoupling and ligand mutation with our protocol, should not add dramatically to the simulation time required for our protocol.