Cryo-EM reconstruction of R-IscB-VR4 bound to ssRNA revealed two conformational states, seed-duplex and full-duplex. (A) Domain organization of R-IscB-VR428 with ssRNA-cleaving mutations marked. Color scheme is consistent throughout Figure 4. (B) Diagram of hybridization formed between guide RNA and target ssRNA. (C-E) Seed-duplex (top) and full-duplex state (bottom) illustrated in domain organization (C) and in cryo-EM densities their corresponding structural models (D-E). Note the differences in HNH domain location, guide RNA direction, and gRNA/tDNA length. (F) Overlay of the fully-duplexed R-IscB-VR4 structure and the AlphaFold3-predicted R-IscB model. Residues beyond N434 could not be modeled in the density due to high mobility. (G) Representative BLI traces of R-IscB-VR4 and its M402A/D403A variant binding to an ssRNA substrate. (H) Representative association rate constants of R-IscB-VR4 RNP and its variants. A complete table of ka, kdis, and Kd is provided in Table S2.
#IscB #ssNA targeting #cryo-EM
2 states: seed duplex (~10 bp) → full pairing (15 bp)
HNH roadblock: blocks #heteroduplex extension, active site hidden, occludes RuvC
Structure-guided R-IscB:
M402A/D403A → 40× rate, 22× affinity
F196H → 2× rate
ssNA = native function
www.biorxiv.org/content/10.6...
