Our simulations of the open conformational state (Figure ) showed no obvious differences between the three different enzymes, suggesting that the relative conformational stability of this state was not notably perturbed as a result of the substitutions that separate each enzyme. However, our simulations of the wide-open state (Figure ) did however clear differences in the relative stabilities of for the different enzymes. We note that while we have observed the wide-open conformation of the WPD-loop as a rare event in simulations of wild-type YopH , there is no available crystal structure of the wild-type enzyme in this conformation. We therefore generated independent starting structures for our wide-open wild-type YopH simulations in silico, using both the Chimera 3 and 2 structures as models for this conformation of the WPDloop. Reassuringly, these two different wide-open starting structures of the wild-type YopH WPDloop sampled similar distributions, and were observed to be approximately equally stable, independent of starting conformation (Figure By combining our RMSD analysis presented above with the available crystallographic data , we can propose the following population shifts between wild-type YopH and Chimeras 3 and 2. First, the five substitutions that separate wild-type YopH and Chimera 3 are responsible for destabilizing the closed WPD-loop conformational state, with similar instability observed for Chimeras 3 and 2 (which are separated by a further two substitutions). Second, the two substitutions that separate Chimeras 3 and 2 are responsible for the observed increased stability in the wide-open conformation of Chimera 2. This increased propensity to populate the catalytically inactive wide-open conformation will clearly contribute to the impaired catalytic activity of this variant, both compared to wild-type YopH, and compared to Chimera 3 .