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- EMDB-80301: Cryo-EM structure of TasH-tigRNA-MM1 dsDNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-80301
TitleCryo-EM structure of TasH-tigRNA-MM1 dsDNA complex
Map data
Sample
  • Complex: Putative nuclease
    • Protein or peptide: Putative nuclease
    • RNA: RNA (37-MER)
    • DNA: DNA (38-MER)
    • DNA: DNA (38-MER)
  • Ligand: MAGNESIUM ION
KeywordsPutative nuclease / ANTIVAL PROTEIN/RNA/DNA / ANTIVAL PROTEIN-RNA-DNA complex
Biological speciesSalicola phage CGphi29 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsZhang H / Liu Z
Funding support China, 1 items
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, 2026-
Header (metadata) releaseAug 26, 2026-
Map releaseAug 26, 2026-
UpdateAug 26, 2026-
Current statusAug 26, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_80301.png

Downloads & links

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Map

FileDownload / File: emd_80301.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.69 Å/pix.
x 320 pix.
= 221.44 Å		0.69 Å/pix.
x 320 pix.
= 221.44 Å		0.69 Å/pix.
x 320 pix.
= 221.44 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.692 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.172
Minimum - Maximum-0.2436162 - 0.61606437
Average (Standard dev.)0.00026803758 (±0.020525988)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 221.43999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_80301_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_80301_half_map_2.map
Projections & Slices
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Sample components

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Entire : Putative nuclease

EntireName: Putative nuclease
Components
  • Complex: Putative nuclease
    • Protein or peptide: Putative nuclease
    • RNA: RNA (37-MER)
    • DNA: DNA (38-MER)
    • DNA: DNA (38-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Putative nuclease

SupramoleculeName: Putative nuclease / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Salicola phage CGphi29 (virus)

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Macromolecule #1: Putative nuclease

MacromoleculeName: Putative nuclease / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Salicola phage CGphi29 (virus)
Molecular weightTheoretical: 40.031242 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNKQVLKEQA SHCEITGAPL AGLPELVDVD RITERFQGGT YTPDNTRVLT PRAHMERHGI LRERDQWLEE LKAMMDDRAQ TMKVVMKMN NQLLAYQRQT DHARQSTEQF LQDTLDASNK RLAQIDREVT KHIKHAKDPL AQAAMGVPGV GPITVAGLQT Y VDLEKAKS ...String:
MNKQVLKEQA SHCEITGAPL AGLPELVDVD RITERFQGGT YTPDNTRVLT PRAHMERHGI LRERDQWLEE LKAMMDDRAQ TMKVVMKMN NQLLAYQRQT DHARQSTEQF LQDTLDASNK RLAQIDREVT KHIKHAKDPL AQAAMGVPGV GPITVAGLQT Y VDLEKAKS ASALWAYIGI DKPSHDRYTK GEAGGGNKTL RTMVWNMANS MIKNRKCPYR TVYEQTKERL AVSEKVTKSR NT QGQLIEC AWKDTKPSHR HGAALRAVMK HFLADYWFVG RELAGLDTRP LYVQEKLGHT GIVQPQERGW EWGGSWSHPQ FEK GGGSGG GSGGSAWSHP QFEKNLYFQS GSHHHHHH

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Macromolecule #2: RNA (37-MER)

MacromoleculeName: RNA (37-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Salicola phage CGphi29 (virus)
Molecular weightTheoretical: 11.948239 KDa
SequenceString:
AGUCAUUCCG UUAAAGACAA CCACGGAGAC GAAGCGA

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Macromolecule #3: DNA (38-MER)

MacromoleculeName: DNA (38-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Salicola phage CGphi29 (virus)
Molecular weightTheoretical: 11.615465 KDa
SequenceString:
(DC)(DC)(DC)(DT)(DA)(DA)(DG)(DG)(DC)(DA) (DA)(DA)(DT)(DC)(DC)(DG)(DT)(DT)(DA)(DC) (DG)(DT)(DC)(DT)(DC)(DC)(DG)(DT)(DG) (DT)(DT)(DA)(DC)(DA)(DG)(DG)(DA)(DC)

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Macromolecule #4: DNA (38-MER)

MacromoleculeName: DNA (38-MER) / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Salicola phage CGphi29 (virus)
Molecular weightTheoretical: 11.775562 KDa
SequenceString:
(DG)(DT)(DC)(DC)(DT)(DG)(DT)(DA)(DA)(DC) (DA)(DC)(DG)(DG)(DA)(DG)(DA)(DC)(DG)(DT) (DA)(DA)(DC)(DG)(DG)(DA)(DT)(DT)(DT) (DG)(DC)(DC)(DT)(DT)(DA)(DG)(DG)(DG)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 49.97 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 86329
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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