Comparing SepTop to other alchemical binding free energy methods may not be advisable for some systems. If ligands in a dataset are structurally very diverse and fall outside the scope of standard RBFE approaches, in addition to RBFE methods like SepTop, binding free energies can be calculated using standard absolute binding free energy calculations. For some targets, however, ABFE calculations are expected to be extremely difficult or nearly impossible without extraordinary long simulations or new algorithmic developments. For example, a protein may undergo a substantial and slow conformational change on ligand binding (e.g. HIV-1 protease, which has a large flap motion on inhibitor binding), or a protein may undergo a change in protonation state on ligand binding (e.g. BACE1, here). In such cases, a normal ABFE calculation which simply removes the ligand from the binding site would leave the protein in an metastable "unbound" state unless simulations are extremely long (in the case of conformational change) or special algorithms are employed (in the case of protonation state changes). In more detail, the two catalytic aspartates in BACE1 were both ionised in the protein-ligand bound state, but that is likely to change to a single ionised and one neutral Asp in the apo state. Thus, we deliberately avoid testing ABFE calculations on BACE1 as we wish to avoid problems caused by an incorrect protonation state for the unbound state. Likewise, we also do not compare to standard RBFE calculations for the scaffoldhopping transformations considered here, as these are simply disallowed by standard RBFE calculations, making comparison impossible. In general, we expect SepTop to be computationally more efficient than ABFE and slightly less efficient than standard RBFE (on transformations where RBFE is applicable).