Multiple approaches to selecting stable atoms for Boresch-style restraints have been reported, with some selection criteria being similar across implementations while other criteria differ. In these studies, equilibration simulation ranging from 1 ns 34 to 20 ns were performed to help identify stationary points in the protein and ligand. Different approaches for identifying these stable structural elements were explored, such as selecting sets of atoms with the most frequent hydrogen bond and salt bridge interactions during MD, looking for buried residues with likely low mobility by calculating the minimal solvent-exposed surface Figure . Thermodynamic cycle for SepTop for computing the free energy difference between two ligands in the binding site (F). The non-interacting dummy ligand (green) is inserted into the binding site and restrained using orientational (Boresch-style) restraints 29 ) (A). The van der Waals (vdW) interactions of the green ligand are turned on and the magenta ligand is restrained (B) and in the next step the electrostatic interactions of the green ligand are turned on while at the same time the electrostatics of the magenta ligand are turned off (C). Then, vdW interactions of the magenta ligand are turned off while at the same time releasing restraints on the green ligand (D). Lastly, the restraints of the now dummy magenta ligand are released analytically and the ligand transferred into the solvent (E). The free energy difference between the ligands in the solvent was obtained separately by running either two absolute hydration free energy calculations or a relative hydration free energy calculation using a SepTop approach.