5.1. We first tested SepTop on the well studied TYK2 dataset as a sanity check. The TYK2 ligands that we investigated here only differ by small R-group changes which allowed us to compare SepTop with standard RBFE to ensure results are reasonable on a system where we can obtain correct binding free energies for the model with a well-established method. Using the same input structures and force field parameters as in a previous study, SepTop and standard RBFE using a non-equilibrium switching protocol gave similar relative binding free energies for three transformations in the TYK2 system. A low cycle closure error of 0.0 ± 0.5 kcal mol -1 (SepTop ϵ-HREX Figure ) suggests that ∆∆G estimates were well converged.