Given our EVB results implied that the catalytic differences observed between the Chimeras and wild-type YopH were linked to the conformational sampling of the WPD-loop, we decided to explore the conformational space available to each PTP and compare the relative stabilities of the different conformational states. As there are no crystal structures available of Chimeras 2 and 3 with a closed WPD-loop conformation, we used two strategies in order to generate structures of the Chimeras with a closed WPD-loop conformation. The first strategy was to mutate (in silico) the wild-type YopH closed loop structure to become Chimeras 2 and 3 and perform MD simulations starting from this conformation, which will be described later. The second strategy employed was to perform targeted MD (tMD) simulations starting from the "wide-open" WPD-loops from both conformations. Using tMD, we slowly "steered" the WPDloop of a given Chimera from the "wide-open" to the "closed" WPD-loop conformation over the course of an MD simulation. To do this, we used the wild-type YopH closed WPD-loop crystal structure as a reference structure, and applied a restraint to the backbone RMSD atoms of the WPD-loop to enforce a slow transition between the two states. The results of our tMD simulations for both Chimeras are presented in Figure , and for wild-type YopH (as a control) are presented in (Figure ). For all systems, we performed 20 replicas per system, pulling the conformations to the closed WPD-loop over the course of 30 ns. Following this, the pulling force was progressively removed over the course of 5 ns, and 10 ns of unrestrained MD simulations were then performed. Analysis of Figures and shows a large spread of RMSDs across the replicas once the restraints were completely released (the last 10 ns of each replica), meaning that in some replicas the closed WPD-loop conformation was retained, whilst in others the WPD-loop reopened rapidly. Similar observations were also made in the control simulations of wild-type YopH starting from a wide-open conformation (Figures and). To identify the potential causes for why some tMD simulations gave rise to relatively stable closed WPD-loop conformations whilst others did not, we investigated the last 5 ns of each replica.