Fig. 3: Integrity of the BH allosterically regulates the helical transitioning of the RuvC-lid to activate for DNA cleavage.

a Highlighted ribbon representation of the BH (bridge helix, pink), RuvC-H1 (RuvC-helix 1, purple), and RuvC-lid (medium purple) regions within the overall structure of state S4b of FnoCas12a^KD2P shown as a surface depiction. The nucleic acids were omitted for clarity. b Close-up view of the BH, RuvC-H1 and RuvC-lid regions of FnoCas12a^KD2P (state S4b; darker shades) overlaid with the corresponding regions from FnoCas12a^WT (PDB: 6GTG²⁰; lighter shades). In FnoCas12a^WT, both BH and the RuvC-lid have undergone helical transitions, but this is not seen in FnoCas12a^KD2P. c, d Close-up view of panel b rotated 120° about the z-axis relative to (b). c In FnoCas12a^KD2P S4b, K981 of the RuvC-H1 interacts with E1020 of the RuvC-lid that is in the loop form. d In FnoCas12a^WT pre-catalytic state (I4²⁰), both BH and RuvC-lid are in helical form. Several distinct contacts are observed in this state compared to that in S4b: E961 of the BH interacts with K1021 of the RuvC-lid, R964 of the BH with E1020 of the RuvC-lid, and R968 of the BH with E1020 of the RuvC-lid. K981 of the RuvC-H1 forms a distinct contact with N1022 of the RuvC-lid and loses its interaction with E1020 of the RuvC-lid in S4b state of FnoCas12a^KD2P. Distances for these interactions are shown in the figure (see Supplementary Figs. 14 and 15 for additional interactions between BH, RuvC-lid and RNA/DNA). e, f Graphs representing linearization of DNA substrates over time for the FnoCas12a protein variants: e matched (MCH) and (f), mismatched (MM13, mismatch at position 13). (Also see Supplementary Table 11 and Supplementary Figs. 17 and 18 for additional information on total cleavage including nicked and linear species, and cleavage efficiency of the variants with MM2 substrate having a mismatch at position 2). The different time points are shown in minutes: 1 (light blue), 2.5 (gray), 5 (pink), and 15 (light green). Each column represents the average of three independent replications; black diamond markers indicate values for individual replications; error bars denote S.E.M. Variants: Wild-type (WT), K969P-D970P (KD2P), E1020A (EA), K1021A (KA), R964A (RA), E1020A-K1021A (EA/KA). Figures (a–d), and graphs (e, f), were made using ChimeraX⁵⁸ and Microsoft Excel respectively. Raw data and calculations used for generating the graphs are provided as a Source Data file.