In this work, we observed re-hydration of the binding cavity in stage 7 for one of the systems we studied (see Section 3.1.3 for details), but only when we started the simulation of stage 7 from a structure of the system that did not have time to adapt to the cavity created by the decoupling of the trapped water. For that one system, we tweaked our setup to use an equilibrated structure from stage 6 to provide starting coordinates for the simulation of stage 7, after which we did not observe any re-hydration of the binding site in stage 7 anymore. 2.3.3. Avoiding conformational changes of the binding site. One of the key findings of the prior work in this space is that, after a trapped water is removed, the cavity left behind by the trapped water can collapse, i.e., the protein can exhibit a conformational change upon decoupling of the trapped water as the protein's side chains can now occupy the space previously occupied by the trapped water. Such a conformational change in the binding site can push the end states with the interacting and noninteracting trapped water far apart in phase space, which in turn can lead to problems in converging the free energy estimate. To ensure that the binding site's conformation does not change upon decoupling of the trapped water, we applied harmonic Cartesian position restrains on the heavy atoms of the binding site of the trapped water (edge D). The Cartesian position restraints effectively make the stages 4 and 5 of our ABFE thermodynamic cycle origin-dependent, i.e., they are not invariant under translation or rotation.