For our RBFE protocol for the transformation of ligands (Figure ), we selected systems for which the binding affinities of two ligands for a target protein are experimentally known, and one of the two ligands binds to the target protein with exactly one trapped water while the other ligand displaces it. For testing our RBFE protocol, we identified five such ligand pairs (Figure ), one for each of the five target proteins: Thrombin, Factor Xa, Scytalone Dehydratase, Beta-Secretase-1 (BACE1), and Bruton's Tyrosin Kinase (BTK). The RBFEs for the ligand transformations for these proteinligand systems have also been previously calculated by various computational studies.