The initial structure of the active site 10 -obtained from superimposition of the cryo-EM structure of the M5:rRNA complex and that of the solution Xray structure of the N-terminal domain of M5-shows two Mg 2+ separated by only 4.23 Å. They are coordinated by 5 carboxylate groups from protein side chain-3 directly (Glu96, Glu10 and Asp56) and 2 through a water molecule (Asp14 and Asp58). The scissile phosphate is positioned to be in direct contact with only one of the two Mg 2+ ions (Mg 2+ A ), and the oxygen of a water molecule is located in between the two cations (Figure ). This arrangement differs from the typical organization of 2-ion dependant nucleases, where the scissile phosphate usually occupies a bridging position in between the two active site ions. Simulations of a reduced model of the system-comprising M5, the protein cofactor uL18 and a fragment of rRNA terminated by a loop (Figure ), all explicitly solvated in water with 150 mM background KCl-showed that, when starting from the structure with phosphate coordinated to a single ion, the active site structure is unstable and the distance between the two ions increases quickly (Figure ).