25QL

Cryo-EM structure of TasH-tigRNA-MM5 dsDNA complex

Summary for 25QL

• Entry DOI: 10.2210/pdb25ql/pdb
• EMDB information: 80302
• Descriptor: Putative nuclease, RNA (37-MER), DNA (38-MER), ... (4 entities in total)
• Functional Keywords: putative nuclease, antival protein/rna/dna, antival protein-rna-dna complex
• Biological source: Salicola phage CGphi29; More
• Total number of polymer chains: 5
• Total formula weight: 115401.75

Authors

Zhang, H.,Liu, Z. (deposition date: 2026-04-14, release date: 2026-08-26)

Primary citation

Zhou, R.,Zhan, Y.,Sun, Y.,Wang, R.,Wang, T.,Liu, Z.,Shan, Z.,Li, X.,Zhang, S.,Sun, N.,Zhang, H.,Yuan, Z.,Yang, J.
Molecular basis of single-mismatch-induced nuclease-to-nickase conversion in TIGR-TasH.
Nucleic Acids Res., 54:-, 2026

PubMed Abstract: Tandem interspaced guide RNA (TIGR)-Tas systems are a distinct class of RNA-guided double-stranded DNA nucleases that employ dual-spacer guide RNAs (tigRNAs) for PAM-independent target recognition. A single mismatch between the tigRNA and target DNA can convert Salicola phage CGphi29 (Sp)TasH from a double-strand nuclease into a nickase in a position-dependent manner, but the molecular basis underlying this functional switch remains unknown. Here, we combined biochemical analyses and cryo-electron microscopy to investigate tigRNA maturation and mismatched target recognition by the Nop domain of SpTasH. We show that the Nop domain is required for pre-tigRNA processing and stabilizes the mature tigRNA through extensive interactions, thereby establishing a cleavage-competent ribonucleoprotein complex. Structural analyses of SpTasH complexes bound to substrates containing single mismatches reveal that a mismatch at the 5'-most position of spacer A is readily accommodated through Nop domain-mediated stabilization of the spacer-target heteroduplex. In contrast, a mismatch proximal to the cleavage site destabilizes the heteroduplex, preventing recruitment of the corresponding HNH domain, thereby converting the complex into a nickase. Together, these findings establish the structural basis for position-dependent mismatch recognition and reveal how Nop domain-mediated tigRNA-target stabilization enables differential responses to mismatches, providing a foundation for engineering TIGR-Tas systems for genome-editing applications.

PubMed: 42531075
DOI: 10.1093/nar/gkag743

Experimental method

ELECTRON MICROSCOPY (3.02 Å)