Molecular Dynamics (MD) simulations of wild-type YopH and Chimeras 2 and 3 were performed in the phospho-enzyme intermediate state (the starting state for the second rate-limiting hydrolysis step). We have previously performed MD simulations on the closed and open WPD-loop states of wild-type YopH, and, in this work, we used these prepared systems to simulate wild-type YopH (and prepared the structures of Chimeras 2 and 3 in a consistent manner). Briefly, the Amber ff14SB force field and TIP3P water model were used to describe protein and water molecules, with simulations performed using AMBER 18 . Simulations were performed at constant temperature and pressure (298 K, 1 atm), with a 2 fs time step. All systems were equilibrated for production MD simulations using the same protocol, which is described in full in the Supporting Information. Each production simulation was 500 ns in length, and performed in 15 replicates. The convergence of these simulations is shown in Figures .