11IC

IscB-TID truncated and wRNA bound to Target ssRNA

Summary for 11IC

• Entry DOI: 10.2210/pdb11ic/pdb
• EMDB information: 75710
• Descriptor: IscB, wRNA (233-MER), Target ssRNA, ... (4 entities in total)
• Functional Keywords: iscb, cas9, hnh, crispr-cas, rna binding protein, rna binding protein-rna complex, rna binding protein/rna
• Biological source: synthetic construct; More
• Total number of polymer chains: 3
• Total formula weight: 134515.30

Authors

Xu, C.,Yang, Q.,Niu, X.,Ke, A. (deposition date: 2026-02-25, release date: 2026-06-03, Last modification date: 2026-07-01)

Primary citation

Xu, C.,Yang, Q.,Niu, X.,Ke, A.
Structure basis for single-strand nucleic acid targeting by IscB and variants.
Nucleic Acids Res., 54:-, 2026

PubMed Abstract: Transposon-encoded IscB was defined as the evolutionary ancestor of CRISPR-Cas9. This compact RNA-guided endonuclease has since been engineered for genome-editing applications. We previously repurposed IscB and related Cas9s as efficient RNA editors by removing their double-stranded DNA (dsDNA) recognition module, the target-adjacent motif (TAM)/protospacer adjacent motif-interacting domain. Here, we report four cryo-electron microscopy structures of IscB, with or without TAM-interaction domain (TID), in complex with single-stranded nucleic acid (ssNA) targets. Structures reveal that, regardless of TID presence, IscB engages ssNA using the same mechanism. IscB initially facilitates formation of a 10-nt seed duplex with ssNA; further base-pairing is blocked by an alternatively positioned HNH nuclease that acts as a roadblock. In this intermediate state, neither HNH nor RuvC is competent for target cleavage. Only upon full duplex formation is the HNH roadblock dislodged by the duplex extension between guide RNA and ssNA. HNH and RuvC nuclease active sites become exposed as the result. A similar set of conformational rearrangements likely governs IscB activity during dsDNA target interrogation. Guided by the structural and mechanistic insights, we introduced mutations to either improve ssNA binding or ease HNH dislodging. Both approaches improved the RNA-targeting efficiency of IscB in vitro and in human cells.

PubMed: 42328786
DOI: 10.1093/nar/gkag607

Experimental method

ELECTRON MICROSCOPY (2.63 Å)