Updated project metadata. Here, we present the crystal structure of the catalytic domain of M5 bound to two magnesium ions, which reveals the role of each catalytic site acidic residue in metal binding. We further use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the possibility of structural rearrangements that would allow cleavage of the 5’-strand, and use small-angle X-ray scattering (SAXS) to study the M5 binding to the pre-5S rRNA substrate. This study provides new details on the M5 mechanism used for cleavage of the dsRNA substrate, which proceeds via two metal ions and structural rearrangements of both M5 and the pre-5S rRNA substrate.