We quantified the location and dynamics of contacts between M5 and the rRNA, on long (4 replicas of 500 ns) standard MD simulations, which we compared with two 500 ns replicas using the REST2 Hamiltonian Replica Exchange enhanced sampling methodology. This technique allows to accelerate sampling of biomolecular conformations by using replicas with scaled interactions-intramolecular and intermolecular-for M5 and rRNA, which mimic a local temperature increase. To avoid spurious disruption of the active site during these simulations, the distance between both Mg 2+ and their non-water ligands was restrained (see Methods).