The overlap of forward and reverse work distributions provides a helpful further diagnostic for the NEQ approach. Having sufficient overlap of the distributions is necessary, but not sufficient for obtaining unbiased free energy estimates; as we found in Section 5.3.2, in some cases, inadequate sampling leads to a false appearance of good overlap. Slow degrees of freedom also often lead to poor overlap, which results in a high analytical error estimate. In addition, bimodal work distributions and sudden changes in work values can be a warning sign that sampling might be complex and slow. Calculating the cross-correlation between side chain dihedral angles in the protein and the non-equilibrium work values can help to diagnose problems. Instead of dihedral angles one could also choose other metrics for protein motion, such as side chain RMSD values. It is important to mention however, that analysis of correlations is prone to false positives -both for correlations between DOF and dH/dλ curves in the EQ approach and DOF with work values in the NEQ approach. It should therefore be applied with caution and the use of replicates for cross-correlation.