The legs L30 à L2, L2 à L* and L* à L30 generate an average difference of 0.17 kcal/mol within the closed cycle (L30 à L2 à L* à L30, Figure ) between the calculations and the experiments. As we have stated previously , a hysteresis value of 0 is a necessary but not sufficient condition for convergence of predictions. An overall hysteresis near 0 for a closed cycle is not necessarily an indication of convergence for each leg; it may be a result of cancellation from individual legs. It is understandable that extending an alkyl group into a hydrophobic binding pocket (Figure ) makes the binding more favourable, of which TIES and experiment agree with each other directionally. It is likely that the force field and/or the sampling attribute to the differences between the calculations and the experimental measurement, but we cannot pin down the source of the apparent overestimation from the simulations. Ligand pairs sharing the common change. 50 pairs have been identified which involve a common variation between the two compounds in the pairs, changing from a -CH group to -C-CH-(CH3)2 at the hydrophobic site. This is the same change as that between L30 to L2 (Figure ). Simulations for the pair get an overestimation of 1.28 kcal/mol for the binding free energy difference: -4. of -3.82 kcal/mol is obtained from TIES calculations, while an average of -2.42 kcal/mol from experimental data. A recent study has proposed a combination of machine learning (ML) and alchemical free energy calculation to improve the accuracy of free energy predictions . The ML method is used to learn the differences between the calculations and the experimental data and derives a correct term which brings the two closer. Such an approach may identify and subsequently correct the discrepancies as observed here. It should be noted that the ML approach used in the study only learns the differences between the calculations and experiments, with no attempts to identify the source of the discrepancies. Studies have shown that a large set of typical chemical modifications lead to a nearly normal distribution of binding energy differences . For individual modifications, however, a wide and non-Gaussian distribution has been observed for experimental binding free energies from a large number of independent measurements, which indicate the assumption of Gaussian statistics may not be justified . If, on average, these 50 compound pairs have the same discrepancy as that in L30 à L2 transformation, the systematic differences might be collectively "corrected". If we apply an offset and shift up all of the TIES result by 1.28 kcal/mol, the calculations agree well with the experimental data, with an MSE reducing from -1.40 kcal/mol to -0.12 kcal/mol (Figure ). This offset could be associated with incomplete sampling of the protein conformational movement that is required to accommodate the change from a -CH group to -C-CH-(CH3)2 in the region of the DFG loop. DFG loop sampling is known to require longer timescales than studied here .