To implement the various restraints required to simulate both the ABFE and RBFE thermodynamic cycles, we modified the GROMACS topology files of the complexes, details of which are described in the Supporting Document Section S1.3. The topology files for all the ABFE and RBFE systems can be found in the GitHub repository associated with this paper, waterNES. 2.6.2. Energy minimization and equilibration. All MD simulations were run using GROMACS 2022.1 simulation package. Water molecules in the system, except the trapped water, were constrained using SET-TLE algorithm. The atoms of the trapped water were constrained according to the procedure described in Supporting Document Section S1.3 (it could not be constrained with SETTLE because, in GROMACS, SETTLE cannot be applied to two different kinds of "molecules"; in this case, the normal water and the (separately treated) trapped water). Coulomb and vdW interactions were calculated for a 10 Å cut-off radius and long-range Coulomb interactions were calculated using Particle Mesh Ewald (PME) with a Fourier grid spacing of 1 Å. A time step of 2 fs was used in MD simulations.