Overall, the interface between M5 and rRNA is stable, even in simulations with enhanced sampling, with a significant fraction of native contacts (> 70 %, Figure ) at the interface preserved all along the simulations. Unsurprisingly the main structural fluctuations can be assigned to flexible RNA and protein loops, often distant from the interface (Figure ). We quantified at the residue-level the change in interfacial contacts with respect to the initial structure using the average value of the signed distance in the space of contacts ∆ i relative to the initial structure (see Methods) (Figure ), and characterized the fluctuation in contacts along the simulations with the per-residue variability of contacts f i (see Methods). In nonaccelerated simulations, the largest changes and flexibility are observed around the U23 RNA base-which is inserted in between the two M5 protein domains-and for the C116 nucleotide, which interacts with the residues around Lys91, in an unstructured loop of M5 N-terminal domain. Large changes in contacts are also found for A55 in the domain β, which changes interaction patterns with the residues around Met136, at the foot of M5 C-terminal domain α7-helix. Three distinct conformations of the interface were identified by performing clustering in the contact space (Figure ), without large conformational changes from the initial experimentally-derived structure. Rather, we observe small side chain rearrangements -e.g. formation of additional salt bridge interactions between protein side chains and RNA phosphate backbone or insertion of the U23 RNA base deeper in the groove between the two protein domains.