Fig. 3: Molecular architecture of HrAgo1 bound to a guide RNA.

a Schematic diagram of the domain organization of HrAgo1. N, N-terminal domain; L1 and L2, linker domains; PAZ, PIWI-ARGONAUTE-ZWILLE domain; MID, Middle domain; PIWI, P-element induces wimpy testis domain. b Schematic representation of the HrAgo1-bound guide RNA. Structurally ordered residues are colored red, while disordered residues are colored gray. c Cryo-electron microscopic (cryo-EM) density map of HrAgo1 bound to a guide RNA Cartoon, colored according to individual domains. The unmodeled 3’-end guide RNA density is represented as a transparent surface. d Cartoon representation of the overall structure of the HrAgo1-guide RNA complex. e All-against-all structure comparison of selected Argonaute proteins. Source data are provided in the Source data file. f Close-up view of the HrAgo1 catalytic site aligned to that of other representative Argonaute proteins. g Close-up view of the HrAgo1 guide RNA 5’-end binding site in the MID domain aligned to that of other representative Argonaute proteins. h Efficient HrAgo1-mediated RNA cleavage requires a guide RNA with a 5’ phosphate and an intact catalytic site. HrAgo1 was incubated with ssDNA or ssRNA guides and Cy5-labeled ssRNA targets. DM: HrAgo1 catalytic mutant with D585A and E623A substitutions. i HrAgo1 mediates RNA-guided RNA cleavage at temperatures ranging from 9 °C to 71 °C. HrAgo1 was incubated with ssRNA guides and Cy5-labeled ssRNA targets. For (h) and (i), Cy5-labeled cleavage products were resolved on a denaturing (7 M urea) polyacrylamide gel and visualized by fluorescence imaging. In (h) and (i), both uncleaved ssRNA targets are 45nt. The results of the cleavage assays were confirmed by at least three repetitions.