Overall, in a shortened RBFE protocol (Figure ), we simultaneously apply the harmonic restraint on the trapped water, solvent repulsion, and position restraints on the binding site (see Section 2.3 for details) in edge M. Thereafter, we decouple the trapped water using an . Thermodynamic cycle to calculate the complex leg of the relative binding free energy (RBFE) between two ligands, where one ligand (green) binds to the protein with a trapped water while the other ligand (purple) displaces it . The red circles in stages 1 through 4 and in the starting stage of edge A represent interacting trapped water, whereas the faded red/ magenta circles in stages 5, 5', 6', 7', 1', and in the end stage of edge I represent non-interacting water. The dashed black circles around the trapped water in stages 3 through 5 and in stages 5' and 6' represent solvent repulsion. The black crosses in stages 2 through 5 and in stages 5' through 7' represent position restraints applied to different parts of the system -the trapped water and the protein's binding site. For an easy nomenclature, the stages and edges for the purple ligand are denoted with a ' superscript for the corresponding stages and edges for the green ligand in the ABFE thermodynamic cycle (Figure ). We have named the end state of edge H as stage 1' because it represents the unrestrained binding mode of the purple ligand and its corresponding stage in the ABFE thermodynamic cycle is denoted by stage 1, i.e., the unrestrained binding mode of the green ligand.