At this time point, no restraints would have been applied to the system for at least 5 ns, meaning the structure would have had time to somewhat "relax". Our analysis revealed the requirement for the P-loop residue R409 to be coordinating the phosphate group in order to facilitate proper closure of the WPD-loop (Figure ). That is, R409 can adopt two major conformations as depicted in Figure , in which the non-coordinated conformation sterically blocks productive loop closure.