Here, we have studied systems with exactly one trapped water in focus. our separation of states protocol can be extended to study systems where a small network of trapped waters might play a role in ligand binding and different congeneric ligands might perturb this network differently. We believe that, with minor changes in our post-processing techniques and with the general framework of our RBFE protocol, RBFE calculations involving small water networks can easily be performed. For example, multiple trapped waters can be restrained in the restraining edge of the RBFE thermodynamic cycle (edge H, Figure ), and all of these trapped waters can be simultaneously decoupled/coupled in a single NES simulation (edge E, Figure ) before transforming the ligand (edge K, Figure ).