Lastly, we tested the performance of SepTop on charged ligands with diverse scaffolds binding to β-secretase 1 (BACE1). BACE1 has been used as a benchmark system for free energy calculations. Different scaffold series of BACE inhibitors have always been treated separately, meaning that separate RBFE maps were run for each scaffold. Here, we tested SepTop on ligand transformations both within different scaffold series, but also across different series. This dataset and those scaffold hopping transformations would be very challenging using standard RBFE methods making this a good test case for the domain of applicability of SepTop. We ran calculations for three different ligand series, the amide series, a spirocyclic series and a biaryl series (Figure ). Different series conserve the interaction with the catalytic aspartate dyad, while the rest of the scaffolds are diverse. Especially the amide series, having a longer linker, extends into the P3 pocket, displacing some water molecules that are present in the two other series. We chose six ligands per series and performed calculations both within each series as well as across different series.