Most of the observations made for the ABFE thermodynamic cycle are also important for RBFE calculations. The importance of restraining the trapped water remains since edge E is identical in the ABFE and RBFE thermodynamic cycles. It is paramount that we prevent the re-hydration of the binding site once the trapped water is decoupled because we want to grow the new ligand in a dry cavity. We also need to ensure that the binding cavity does not collapse upon removing the trapped water since that would make growing the new ligand more complicated. In contrast to the ABFE cycle, we are however expecting that the release of the binding site restraints will consist of a shorter path in phase space, since the larger ligand should fill out most of the space left behind by the removed trapped water. 2.5. Selected systems. To test our ABFE protocol (Figure ), we selected the 13 systems previously studied by Ge et al., since our efforts are directed toward optimizing the separation of states protocol originally introduced in that study. The 13 systems were originally taken from a previous study by Barillari et al., which focused on classifying conserved and displaceable water molecules across a large number of proteins/protein-ligand complexes. The 13 systems studied here consist of a Bovine Pancreatic Trypsin Inhibitor protein (BPTI; PDB ID: 5PTI ), and three proteinligand complexes of each of Trypsin (PDB IDs: 1AZ8, 31 1C5T 32 and 1GI1 33 ), Factor Xa (PDB IDs: 1LPG, 34 1F0S 35 and 1EZQ ), HIV-1 Protease (PDB IDs: 1EC0, 36 1EBW 37 and 1HPX ), and Scytalone Dehydratase (PDB IDs: 3STD, 39 7STD 40 and 4STD ) target proteins. For each target protein, the trapped water molecule of interest is located at the same location in the binding site of the protein.