ions, but note that the specific choice of the force field is here irrelevant, since restraints maintain the active site structure around the ions. In addition, extensive exploration of the complex conformational space was performed using a Hamiltonian replica exchange scheme to accelerate sampling, namely Replica Exchange with Solute Tempering (REST2), scaling being applied to a subset of atoms comprising the RNA and M5, as well as the 2 Mg 2+ ions and 102 Na + ions to keep a neutral charge in the rescaled region. 32 replicas were used with scaling factors λ ranging from 1 to 0.731707, thus covering an effective temperature range for rescaled atoms of 300 K to 410 K. Exchange was attempted every 2 ps, which yielded an average exchange rate of ∼20 %. This simulation was replicated using random initial velocities.