In SepTop we obtain the relative free energy difference between two ligands in the binding site by running what is essentially two ABFE calculations at once in opposite directions. The thermodynamic path for the transformation of one ligand into the other ligand in the binding site (∆G site ) is shown in Figure . To obtain the relative solvation free energy, ∆G solvent , we perform two absolute hydration free energy calculations if all ligands are neutral. If, on the other hand, ligands have the same non-zero charge we use a SepTop protocol in the solvent leg in order to preserve the net charge in the system (see Section 4.3).