6.5. Current limitations of SepTop. In theory, transformations performed using the SepTop approach are not restricted by structural similarity of the ligands and ligand binding modes, however, there might be limits to the domain of applicability of the method. We expect slow convergence and biased results if the two ligands bind to different conformations of the protein and if transformations between those conformations happen on timescales longer than the simulation run time. Slow convergence can also be expected if one ligand displaces buried water molecules that are not displaced in the presence of the other ligand, especially if the entry/exit of that water molecule is not sampled throughout the simulation. In addition, results are expected to be incorrect if the ligands bind to different protonation states of the protein or if one or more ligands bind covalently, and results may be slow to converge if the ligands bind in disparate regions of the binding site.