In our RBFE calculations, we obtained sufficient overlap between the sampled phase spaces by the unrestrained and restrained states, in both the equilibrium free energy calculations, as well as in the forward and reverse NES transformations for the trapped water decoupling and for the ligand transformation. This resulted in ) of the system. The nearest solvent water exchanges with the trapped water at around 3 ns during the simulation. (Right) Phase space overlap matrix for the equilibrium free energy calculation comprising of two states of the BTK system: an unrestrained stage, and a restrained stage (stages 1 and 4, respectively, in Figure ). In the restrained state, the harmonic restraint on the trapped water, the solvent repulsion term, and the binding site position restraints are in place. The overlap matrix shows that we obtained good phase space overlap between the unrestrained and the restrained states, even though the trapped water was exchanged with a solvent water during the simulation of the unrestrained stage, because we re-mapped the trajectory of the unrestrained stage to identify the exchanged solvent water as the new trapped water upon exchange (see Section 2.3.1 for details).