When initiating simulations of the unliganded SeHisA(dup13-15/D10G) variant starting from these loop 1 closed conformations, we did not observe any opening of loop 1. This is in contrast to our simulations of the wild-type enzyme, where we sampled open conformations of this loop when we started from the unliganded closed conformation observed in PDB ID: 5A5W, removing the ProFAR substrate. This provides evidence, in addition to the discussion in ref. , that the elongation of loop 1 heavily stabilizes this PRA-active conformation. This is further supported by examining the root mean square fluctuations of all Cα atoms in our simulations of these two enzymes (Figure ), where we observe that loop 1 is more flexible than either of loops 5 or 6 in simulations of the wild-type enzyme, but has reduced flexibility in simulations of the SeHisA(dup13-15/D10G) variant. ).