For each of the ABFE and RBFE systems, starting from the solvated structure, we performed an energy minimization for 5000 steps using the steepest descent algorithm. Then, we equilibrated the system in an NVT ensemble for 10 ps, stabilizing the temperature of the system at 298.15 K using the stochastic dynamics integrator and an inverse friction constant of 2 ps. In the NVT equilibration, we restrained each of the heavy atoms of the protein and the ligand using a harmonic position restraint with a force constant of 1000 kJ mol -1 nm -2 . Finally, we performed a 100 ps equilibration of the system in the NPT ensemble, by coupling the system to the Parrinello-Rahman barostat in an isotropic pressure coupling of 1 bar using a time constant of 1 ps. During the NPT equilibration as well, each of the heavy atoms of the protein and the ligand were restrained using a harmonic position restraint with a force constant of 1000 kJ mol -1 nm -2 . Full details of the input parameters for the simulations (GRO-MACS mdp files) can be found in the GitHub repository waterNES. 2.6.3. Equilibrium free energy simulations. For simulation of the individual stages of the ABFE and RBFE thermodynamic cycles, three different λ schedules (Tables and), scaling the harmonic restraint on the trapped water, the solvent repulsion term, and the position restraints on the binding site, were added to the GROMACS input parameters file for each stage (mdp files), details of which are described in Supporting Document Section S1.1. Simulations of all the stages of the thermodynamic cycles were run independently and were started from the structure obtained from the procedure described in Section 2.6.2. The stages were then simulated for a 6 ns production run in the NPT ensemble using the stochastic dynamics integrator and the Parrinello-Rahman barostat. The stages with Cartesian position restraints on the binding site used their respective starting structure (i.e. the final structure of the equilibration) as the reference structure for the position restraints.