pointing "in" towards the substrate ProFAR (PDB ID: 3ZS4 ), and a pro-PRA β-hairpin conformation of loop 5, with R143 pointing "in" towards the substrate PRA (PDB ID: 2Y85 ), extrapolating the substrate positioning from the position of the analogous product PRFAR and product analog rCdRP in the respective conformations. Here, we used PriA in its pro-ProFAR conformation as a reference state to calibrate our EVB simulations of the initial ring-opening of ProFAR. The resulting EVB parameters were then used unchanged in all relevant systems.