Our tMD simulations identified that it is indeed possible to form "closed" WPD-loop conformations for both Chimeras, and that this conformation can be stable (at least on short simulation timescales) in these PTP variants. Likewise, wild-type YopH is known to be able to form a closed WPD-loop conformation, as it has been crystalised in that conformation , unlike the two Chimeras . Taken together, this would suggest all enzymes can sample the three major WPD-loop conformational states (closed, open and wideopen) but with altered favourability among the states. We wished to investigate this proposed population shift across the enzymes and used MD simulations starting from each conformational state to do so. That is, each enzyme was simulated using 15 replicas of 500 ns long MD simulations starting from each conformational state. The Cα RMSD of the WPD-loop residues was measured throughout each simulation and was used to generate the probability distributions presented in Figures . In cases where crystal structures of the conformational state to be simulated were not available for a given enzyme, the required residues were substituted in silico, see the Methods for further details. Focusing first on the closed WPD-loop simulations (Figure ), the simulations show the wild-type YopH WPD-loop closed conformation to be the most stable conformation, which samples a narrower and smaller distribution of RMSD values compared to Chimeras 3 and 2. For example, 95.7% of all wild-type YopH frames have an RMSD ≤1.5 Å from the crystallographic closed conformation, as compared to 57.4% and 45.8% of frames from simulations of Chimeras 3 and 2, respectively. This measurement is robust to different RMSD cut-offs, for example the percentage of frames that have an RMSD ≤2.0 Å for each enzyme are 99.6%, 81.6% and 75.8% for the wild-type enzyme, Chimera 3, and Chimera 2, respectively. The observation that wild-type YopH has a notably more stable closed WPD-loop conformation is consistent with the aforementioned crystallographic data . Further, visual inspection of the individual RMSD traces used to build the histograms depicted in Figure identifies clear examples of WPD-loop opening in Chimeras 2 and 3 (Figure ), in contrast with the simulations of wild-type YopH were this is notably more rarely observed (Figure ). Given the similarity in the probability distributions and the number of observations, it is unclear if there is a difference in the relative stability of the closed WPD-loop state for Chimeras 3 and 2.