Following from this, PCA analysis on our simulations shows that the active site loops in these enzymes are not rigid, but can sample a range of wide-open conformations with transitions between them, with their conformational flexibility being stabilized by ligand binding (although less so in the bifunctional PriA than the ProFAR-specific SeHisA). In contrast, in TrpF, which binds a smaller substrate and lacks the gripper, the active site loops remain dynamic, in particular loop 6 (Figure ), which samples a range of open conformations even with substrate PRA bound to the active site. Related to this, all HisA variants from the real-time evolution experiment studied here also sample a range of open and wide-open conformations. In this context, however, the single D10G substitution on loop 1 appears to be sufficient by itself to increase the population of the closed conformation sampled during our simulations (Table ), and the highest proportion of closed conformation is observed in simulations of the SeHisA(dup13-15/D10G/Q102A/Q24L/V15[b]M) variant, which has the highest TrpF activity 28 (Table ).