From a methodological point of view, we stress that enhanced sampling techniques were key to uncover these distinct conformational states of the active sites and characterize the associated free energy landscape. This work thus underlines the importance to perform extensive conformational exploration of the reactant state of a reactive complex before mechanistic investigations, echoing in that recent work on different RNA processing systems, ribozymes. Finally, while the present study focused on the cleavage of the 3 ′ end by M5, experiments suggest 9 that the complex then undergoes reorganization to position the same M5 enzyme on the 5 ′ end to perform the second cleavage step. While M5 presumably remains anchored to the rRNA with its C-terminal domain, significant reorganization of the complex has to occur after removal of the 3 ′ end to allow for the needed repositionning. Characterization of this reorganization is a challenge both for experiments and computations that will be tackled in future work.