Extended Data Fig. 4. Structural analysis of Cas9.

a, Left, comparison of Cas9 protein only between 12–14 MM 60 min linear (colour) and 12–14 MM 1-h kinked (grey) models. Right, comparison of Cas9 protein only active conformation (18–20 MM 1 min linear, colour) and kinked pre-active (12–14 MM 60 min kinked, grey) models. While there is no significant conformational change associated between transition from linear to kinked pre-active (root-mean standard deviation (RMSD) between equivalent Cα atoms of 1.904 Å), the change from kinked pre-active to active conformations is associated with a larger conformational change (4.647 Å, most of which occurs within the REC3 domain). b, Close-up view of REC3 conformational changes that occur upon activation, as viewed from one angle. REC3 moves forwards towards the kinked duplex by ~15 Å upon activation and HNH repositioning. c, Schematic representation of Cas9–nucleic acid contacts in the context of 18–20 MM. Residues mutated in SuperFi-Cas9 are denoted by an asterisk. d, Conformations of HNH domain (green) and L1 (gold) and L2 (purple) linkers in the context of Cas9 binary complex (i.e. with gRNA, PDB 4ZT0), Cas9–gRNA complex bound to dsDNA in an inactive conformation (PDB 5F9R), and in the active Cas9 18–20 MM structure presented in this work. Upon activation, HNH is repositioned at the target strand cleavage site, driven by large conformational changes in the L1 and L2 linkers. e, Comparison with the active Cas9 18–20 MM structure presented in this work and previously determined cryo-EM maps (transparent grey) of inactive (left, EMD-3276¹⁴) and active (right, EMD-0584¹⁸) Cas9 bound to on-target dsDNA. The inactive Cas9 has no density for L1 helix at the kinked distal-docked gRNA–TS site, whereas there is clear density for L1 at this site in the active Cas9 cryo-EM map. f, Mapping of residues mutated to alanine in selected high-fidelity Cas9 variants. EvoCas9 (yellow) – M495, Y515, R661, K526. Cas9-HF1 (red) – N497, R661, Q695, Q926. HypaCas9 (blue) – N692, M694, Q695, H698. Residues shared between Cas9-HF-1 and either EvoCas9 or HypaCas9 are shown as orange and purple, respectively.