Fig. 5: Analysis of the BH and RuvC-H1 conformations of different Cas12 and Cas9 families.

a A DALI⁴¹ dendrogram representing the hierarchical clustering of structures of BH (bridge helix) and RuvC domain ( ~ 200 amino acids covering motifs I, II, and III) of Cas12 and Cas9 subtypes, and ancestor proteins. The dendrogram shows five distinct clusters of proteins based on the structural similarities of BH and RuvC domain. PDB IDs with the corresponding proteins are indicated at the tip of each respective leaf. The numerical value above each branch indicates the branch length and longer branches show lower degree of structural similarity. b Structural superposition of FnoCas12a [BH in pink, RuvC-H1 (RuvC-helix 1) in purple, and the remaining part of the RuvC domain in medium purple] with few representative proteins belonging to a cluster (see Supplementary Fig. 23a for related figures). The Z-score, RMSD, and number of equivalent residues aligned (Lalin) based on the preliminary DALI search are shown against each superposition for Cas12 subtypes. Since Cas9 subtypes were manually added, rather than being identified through a DALI search, these parameters are not available for them. Each structure aligned with FnoCas12a is shown in one color, without domain demarcation, and the color matches the highlighting of the associated PDB ID. For Cluster V, BH and the helix from RuvC motif-II of Cas9 that aligns with RuvC-H1 of FnoCas12a are shown for selected structures (see Supplementary Figs. 23 and 24 for the remaining analysis of Cas9’s BH and RuvC domain conformational changes). The PDB IDs and the related structures used in this analysis are listed in Supplementary Table 23. Sequences covering the BH and RuvC domain that were used for generating the dendrogram and the structural comparisons are provided in the Source Data file. The dendrogram was generated using an online tool iTOL⁷³ and structure alignments were made using ChimeraX⁵⁸.