4Z3V 44 * indicates the PDB ID of the complex containing a closely related ligand 2.6. Simulation details. 2.6.1. System topology and structure preparation. For the ABFE calculations for the trapped waters, protein/protein-ligand complex structures were downloaded from the Protein Data Bank (PDB) for the respective PDB IDs listed in Section 2.5. Missing residues or heavy atoms were added using PDB-Fixer, and the protonation states for the amino acid residues of the protein were determined according to the respective experimental pH reported on the PDB webpages of the complexes, using the PROPKA algorithm available on the PDB2PQR web server to estimate the most likely protonation state of each residue at that pH. For the HIV-Protease system 1HPX, protonation states for the two aspartic acids (ASP-25 and ASP-125) in the binding site are known from a previous experimental study, and the protonation states we obtained from the PDB2PQR web server agreed with those from the experimental study. The pKa values for ligands were estimated using Chemicalize utility of ChemAxon, and we used the resulting predicted ligand protonation states, except when otherwise noted. We used OpenMM software package to generate the force field parameters for the protein-ligand complexes. Protein structures were parameterized using the AMBER 14SB force field, and ligands were parameterized using the OpenFF-2.0.0 force field (Sage) and the AM1-BCC charge model implemented in the OpenEye toolkit. We used TIP3P water model 53 to solvate our systems and added 0.2 molar Na + and Cl -to the simulation box.