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- PDB-25ql: Cryo-EM structure of TasH-tigRNA-MM5 dsDNA complex -

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Basic information

Entry
Database: PDB / ID: 25ql
TitleCryo-EM structure of TasH-tigRNA-MM5 dsDNA complex
Components
  • (DNA (38-MER)) x 2
  • Putative nuclease
  • RNA (37-MER)
KeywordsANTIVAL PROTEIN/RNA/DNA / Putative nuclease / ANTIVAL PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesSalicola phage CGphi29 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsZhang, H. / Liu, Z.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: Nucleic Acids Res / Year: 2026
Title: Molecular basis of single-mismatch-induced nuclease-to-nickase conversion in TIGR-TasH.
Authors: Ruimin Zhou / Yancheng Zhan / Yangyue Sun / Runqin Wang / Tongyao Wang / Zhikun Liu / Zhengda Shan / Xuzichao Li / Shuqin Zhang / Nan Sun / Heng Zhang / Zhiyong Yuan / Jie Yang /
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 ...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.
History
DepositionApr 14, 2026Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Aug 26, 2026Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Putative nuclease
B: Putative nuclease
C: RNA (37-MER)
E: DNA (38-MER)
F: DNA (38-MER)


Theoretical massNumber of molelcules
Total (without water)115,4025
Polymers115,4025
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Putative nuclease


Mass: 40031.242 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salicola phage CGphi29 (virus) / Production host: Escherichia coli (E. coli)
#2: RNA chain RNA (37-MER)


Mass: 11948.239 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salicola phage CGphi29 (virus) / Production host: Escherichia coli (E. coli)
#3: DNA chain DNA (38-MER)


Mass: 11606.450 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salicola phage CGphi29 (virus) / Production host: Escherichia coli (E. coli)
#4: DNA chain DNA (38-MER)


Mass: 11784.576 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salicola phage CGphi29 (virus) / Production host: Escherichia coli (E. coli)
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Putative nuclease / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Salicola phage CGphi29 (virus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 49.97 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 315323 / Symmetry type: POINT

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