Rationale behind the design of the ABFE thermodynamic cycle. We chose the restraints used in the ABFE thermodynamic cycle (Figure ) keeping in mind various common sampling challenges that can arise in ABFE calculations of trapped water. We sought to ensure that our choice of restraints not only avoid the common sampling issues, but also result in a computationally inexpensive thermodynamic cycle. In the following sections, we have described the rationale behind using the restraints in our ABFE thermodynamic cycle, how we calculated the free energy contributions of those restraints, and how the restraints paved the way to extend the ABFE thermodynamic cycle into an RBFE thermodynamic cycle where ligand transformations can also be carried out, in addition to the decoupling of the trapped water. 2.3.1. Keeping the trapped water in the binding site. In our ABFE thermodynamic cycle, we apply a restraint on the trapped water (in edge B) to ensure that it stays near its putative location in the binding site, even after its interactions are turned off. The restraint is implemented as a harmonic potential with a minimum at the location of the trapped water in the crystal structure. The location is defined as a virtual site relative to two heavy atoms close to the pocket (see Section S1.3 of the Supporting Document for details). Without the restraint, the trapped water would wander off once its interactions with the protein are made sufficiently weak. The water would then need to find its way back into the binding site in the short NES switches of edge E, which is nearly impossible. As a second consideration, adding a restraint on the trapped water also prevents problems with slow, partially solventexposed water which may infrequently unbind and rebind in stages 2, 3, and 4.