Fig. 4. The HNH active site conformation in state II.

(a) Left: Close-up view of the active site of the HNH domain in state II (pink) with the corresponding cryo-EM map (blue mesh) superposed. A putative metal ion position is marked with green asterisk. Right: Schematic representation of the state II HNH active site. Putative Mg²⁺ ion, water molecules and their interactions are colored grey. Observed H-bonds are blue dashed lines, while red dashed lines designate putative interactions between Cas9 side chains and unmodeled Mg²⁺. Distances between coordinating Cas9 residues and the proposed Mg²⁺ion location are shown in Å. (b) The active site of the homing endonuclease I, HmuI (PDB ID 1U3E) ²⁶. (c) The active site of the HNH domain from A. naeslundii Cas9 (PDB ID 4OGE) ⁹. In (a, left), (b) and (c), catalytic residues and reaction products are shown as pink and blue sticks, respectively, and hydrogen bonds, both observed and putative, are shown as dashed lines. In panels b and c, purple, green and red spheres are Mn²⁺, Mg²⁺ and water molecules, respectively.