Active site structure and fluctuations. The experimentally determined M5 active site structure is similar to that of other RNases reported in the literature, with 2 Mg 2+ ions coordinated by several negatively charged protein side chains, either directly or through water molecules. While the initial superimposition of the cryoEM structure of the complexmutated to prevent reactivity-and the X-ray structures of M5 isolated domains suggested an unusual position of the scissile phosphate, coordinating only one of the two ions, this structure is unstable in simulations. We propose that the scissile phosphate should instead, as in other RNases, be positioned in between the two active site ions, thus effectively replacing the water molecule resolved in the crystal structure of the isolated N-terminal domain. This (small) change being done, the active site structure now closely resembles that of other RNases, in particular the well-characterized RNase H. Yet, substantial differences can be noticed. First, the binding of two active site aspartate residues is mediated by water molecules in M5-in contrast with direct inner sphere coordination in RNase H. In addition, while the presence of an aspartate that simultaneously binds both Mg 2+ ions is often reported to be a key element for 2-ion catalysis, M5 is missing such a feature. Our present work suggests that the active site is stable-provided an adequate force field is used in simulations-and that a reasonable reaction free energy path exist without this feature, which may not be as essential as previously thought.