Figure shows the correlation between experimental free energies of binding and calculated relative binding free energies converted to free energies of binding. Figure in the SI shows the calculated RBFE (averaged over the three repeats) against experimental RBFE (converted from K i using Eq. 2). The error bars show the standard deviation from the three repeats, except for transformations of 11 to 14 and 14 to 15, for which the errors are from MBAR of just one repeat. These transformations converge unreliably with the stanard protocol proposed. No additional protocol optimiation for these transformations was carried out. For the resulting binding free energy in Fig. the mean absolute error (MAE) of 0.43 0.66 0.25 kcal/mol is well below typically expected accuracies of 1 kcal/mol. Furthermore, Spearman's rank correlation (ρ = 0.93 1.00 0.75 ) and Pearson's correlation coefficient (R 2 = 0.93 0.98 0.86 ) show that the RBFE calculations, obtained with existing ligand and protein force fields can predict experimental results well. In addition, the overlap matrices (see SI Fig. and) show sufficient overlap between consecutive λ-windows, suggesting that the free energy estimates obtained with MBAR are reliable. The outliers in Fig. and in the SI Fig. , all (except ligand 3 ) involve transformations with halides, such as fluorine and bromine, which are known to be a challenge for smallmolecule force fields, such as GAFF2. Additionally, transformations involving ligand 3 involve a ring formation or breaking, which are known to be difficult transformations. Overall we standard RBFE calculations with the chosen force fields perform well for serineβ-lactamases.