Our ABFE protocol resolves previously reported sampling challenges arising due to the cavity left behind by the decoupling of the trapped water. In our separation of states ABFE protocol, we applied a series of restraints to solve a major challenge in ABFE calculations of trapped waters, i.e., to stabilize the cavity left behind by the decoupling of the trapped water. Previously, Ge et al. reported two major sampling challenges in simulating protein-ligand complexes with this cavity: solvent flooding of the cavity, and binding site collapse. Due to these sampling challenges, their calculations resulted in large errors in their free energy estimates (Figure ). Our protocol resolves both of these sampling challenges by introducing a solvent repulsion term and protein binding site restraints in our thermodynamic cycle (Figure ).