The magnitudes of the estimated errors prevented us from making a definite classification in six of the 12 mutations studied. One hypothesis is that the larger the alchemical perturbation, the larger the magnitude of error. We therefore examined whether there was a correlation between the number of atoms where the atom type was perturbed during the alchemical transition and the magnitude of error in calculated DDG values (Fig. ). There was a weak positive correlation for both RNAP and DNAG mutations, suggesting that whilst this does play a role, it is not the main driver behind the large errors observed here.