For the NEQ approach we calculated free energy differences using the CFT/BAR method as implemented in pmx. Instead of calculating the mean of three independent replicates, we pooled together all work values from the repeats to estimate one free energy difference. For the uncertainty estimate, we calculated the standard deviation obtained by treating the in-dividual repeats separately. This analysis was performed for decoupling the ligand in the binding site as well as coupling it in the solvent. 4.6. We assessed potential sampling limitations. To help assess sampling problems in this work, we find it helpful to examine several properties relating to free energies, as we will further discuss in Sec. 5. Particularly, we compute the Pearson correlation coefficient between the work values and all side chain (ξ) dihedral angles in the protein using the SciPy package in Python, with ξ angles calculated via GRO-MACS. Additionally, we examine dH/dλ values and their relationship to other degrees of freedom. Here, we do not use dH/dλ values for free energy estimates, but they can be used to do so via TI, and these dH/dλ values provide a view into the potential contributions of specific motions in specific trajectories to binding free energies (since they are measured for individual snapshots, allowing us to identify rearrangements/motions which lead to sudden changes in dH/dλ). We find (Sec. 5.3 and 5.5) that these are a helpful diagnostic.