tion here using the double decoupling method to decouple trapped waters from the proteins' binding sites. They implemented replica exchange thermodynamic integration (TI) in MC simulations, and they used a hard-wall potential at the location of the trapped water to prevent solvent flooding-and a potential collapseof the binding site during the water decoupling. Notably, our ABFEs for the BPTI, Trypsin, and Scytalone Dehydratase systems lie within 2 kcal mol -1 of the corresponding ABFEs from the reference study (Figure ). However, the reference study overestimates the binding affinities of the trapped waters in HIV-1 Protease systems and underestimates them in Factor Xa systems, compared to the binding affinities that we calculated for those two systems.