Scaffold hopping transformations with SepTop on ERα systems. ERα has been studied by multiple groups benchmarking different scaffold hopping approaches and therefore appeared to be a good test system for our method, giving us the possibility to compare SepTop with other methods. Here, we compared SepTop with three different RBFE methods, the alchemical transfer method (ATM), as well as a RBFE method that uses auxiliary restraints for scaffold hopping transformations and lastly FEP+. For these three ligand transformations, different methods converged to similar relative binding free energies (Figure ). Using the same input structures provided as in the auxiliary restraints paper, SepTop converged to the same ∆∆G value within uncertainty as the method using auxiliary restraints in the two edges where calculated values had been reported.