Fig. 1: FnoCas12a^KD2P possesses conformational states with similarities and differences compared to those for FnoCas12a^WT.

a Schematic representation of the domain architecture of FnoCas12a. The domain color scheme is maintained consistently throughout the manuscript: REC lobe [REC1 (light green), REC2 (dodger blue)] and NUC lobe [WED (tan), PI (PAM-interacting, salmon), RuvC (medium purple), BH (bridge helix, pink), RuvC-H1 (RuvC-helix 1, purple), and Nuc (gray)]. Nucleic acids are shown as: crRNA (orange), target DNA [non-target strand (NTS, deep sky blue), target strand (TS, blue), and protospacer adjacent motif (PAM, black)]. b–f Cryo-EM maps of FnoCas12a^KD2P states (b), S1, (c), S2, (d), S3, (e), S4a, and (f), S4b each shown in two orientations. Major conformational difference is in the relative positioning of REC1 and REC2 domains across the different states that enables the opening and closing of the crab-claw structure and the accommodation of the RNA-DNA hybrid in between the two lobes. g–k Corresponding model built to fit the electron density map for states (g), S1, (h), S2, (i), S3, (j), S4a, and (k), S4b. A proposed order for the different states of FnoCas12a^KD2P with respect to binary and pre-catalytic states of FnoCas12a^WT is shown in Supplementary Fig. 10. Between S1 and S4b of FnoCas12a^KD2P, the complex transitions through different amounts of opening of the crab-claw structure, as indicated by the distance between the REC2 and Nuc domains in solid lines (from 12 Å in S1, to 46 Å in S4a and 31 Å in S4b; distance shown is between C-\(\alpha\) atoms of D468 of REC2 domain and V1102 of Nuc domain). Additionally, the separation between the REC1 and REC2 domains also varies in the different states as shown in the dashed lines (from 39 Å in S1, to 77 Å in S4a and 71 Å in S4b; distance shown is between C-\(\alpha\) atoms K85 of REC1 domain and C473 of REC2 domain). See also Supplementary Tables 3 and 4. Figure was made using ChimeraX⁵⁸.