The comparison of results from different methods was challenging due to differences in system preparation in the different studies. We found that the modeling of missing loops (Figure ) and the protonation state of histidine residues (Figure ) had an impact on the predicted binding free energy. We ran calculations with the SepTop approach using an input structure we prepared using OpenEye's Spruce 48 (SI Table input spruce), and with prepared structures used in the studies of Azimi et al. (input ATM) and Zou et al. (input Aux) and obtained very different results (SI Table 1). When using the input structure that had missing loops we had to restrain the position of backbone protein atoms to prevent the protein from drifting apart. This could have potentially impacted the results and partly explain why calculations starting from different input structures did not give equivalent results. For one of the transformations (edge 2d -2e), we performed additional calculations to study the impact of the protonation state of histidine on the results. In one protocol the histidine in the binding site was charged (HIS220, Figure ) and in another protocol HIS220 was neutral, while in one protocol the protonation state was not reported. We found that the protonation state of that histidine in the binding site affected the calculated ∆∆G by ≈ 1 kcal mol -1 . Our finding also shows that depositing prepared protein structures along with a publication is critical for reprehensibility of results. Perturbation cycle in the TYK2 system. For all three transformations, we report the experimental (grey) and calculated relative binding free energies. In the SepTop approach, five different protocols were tested, two without enhanced sampling (EQ and EQ no dihre), one using HREX, and two protocols using ϵ-HREX. In the first three protocols, rotatable bonds were restrained using dihedral restraints while in the fourth and fifth protocol (purple and yellow) no dihedral restraints were applied. The last protocol (teal) shows values obtained from the work of Ge et al. using standard RBFE. SepTop and standard RBFE produced similar results. With SepTop, different protocols converged to the same relative free energies within uncertainty and the standard deviation across trials was lower when using enhanced sampling techniques. Figure . Ligand cycle in the ERα system. For all three transformations, we report the experimental and calculated relative binding free energies. The error estimate in SepTop is the standard deviation calculated across three independent repeats. Overall, different methods gave similar results, however, they did not always converge to the same ∆∆G values.