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- EMDB-65567: Cryo-EM structure of TasH-tigRNA (5G)-dsDNA complex -

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Entry
Database: EMDB / ID: EMD-65567
TitleCryo-EM structure of TasH-tigRNA (5G)-dsDNA complex
Map data
Sample
  • Complex: Cryo-EM structure of TasH-tigRNA (5G)-dsDNA complex
    • Protein or peptide: TasH
    • RNA: RNA (36-MER)
    • DNA: DNA (38-MER)
    • DNA: DNA (38-MER)
  • Ligand: MAGNESIUM ION
Keywordsprotein complex / ANTIVIRAL PROTEIN
Biological speciesSalicola phage CGphi29 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.62 Å
AuthorsZhang H / Liu Z
Funding support1 items
OrganizationGrant numberCountry
Other government
CitationJournal: Mol Cell / Year: 2026
Title: Molecular basis for dual-spacer-guided target cleavage by the TIGR-TasH system.
Authors: Jie Yang / Tongyao Wang / Zhikun Liu / Wenqi Wu / Yangyue Sun / Yancheng Zhan / Shuqin Zhang / Hong Chen / Bin Liu / Caidie Yue / Zhenning Yin / Zhengda Shan / Xuzichao Li / Zhuang Li / ...Authors: Jie Yang / Tongyao Wang / Zhikun Liu / Wenqi Wu / Yangyue Sun / Yancheng Zhan / Shuqin Zhang / Hong Chen / Bin Liu / Caidie Yue / Zhenning Yin / Zhengda Shan / Xuzichao Li / Zhuang Li / Zhiyong Yuan / Hang Yin / Heng Zhang /
Abstract: The RNA-directed programmable nuclease systems, exemplified by the CRISPR-Cas system, have been widely used in genome editing. In contrast to the single-spacer configuration of CRISPR RNA (crRNA), ...The RNA-directed programmable nuclease systems, exemplified by the CRISPR-Cas system, have been widely used in genome editing. In contrast to the single-spacer configuration of CRISPR RNA (crRNA), the guide RNA (tigRNA) of the tandem interspaced guide RNA (TIGR) system features a dual-spacer arrangement, thereby directing the TIGR-associated (Tas) protein to engage both strands of the target double-stranded DNA (dsDNA). Here, we determine six cryo-electron microscopy structures of the Salicola phage TIGR-TasH complex. The central coiled-coil region of TasH mediates dimerization, while the C-terminal nucleolar protein (Nop) domain is able to autonomously process precursor tigRNA. Upon target binding, the dynamic N-terminal HNH nuclease domain is recruited for cleavage through a β-hairpin, which also determines the target preference. More interestingly, the conserved box C motif of tigRNA stabilizes this β-hairpin in an adenine-specific manner, enabling us to rationally design a guide RNA-defined nickase, distinct from conventional protein-based nickase strategies used in genome editing.
History
DepositionJul 26, 2025-
Header (metadata) releaseMar 11, 2026-
Map releaseMar 11, 2026-
UpdateApr 22, 2026-
Current statusApr 22, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_65567.png

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Map

FileDownload / File: emd_65567.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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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
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.17
Minimum - Maximum-0.2807233 - 0.77993476
Average (Standard dev.)0.00034537868 (±0.022669766)
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: #2

Fileemd_65567_half_map_1.map
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Half map: #1

Fileemd_65567_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of TasH-tigRNA (5G)-dsDNA complex

EntireName: Cryo-EM structure of TasH-tigRNA (5G)-dsDNA complex
Components
  • Complex: Cryo-EM structure of TasH-tigRNA (5G)-dsDNA complex
    • Protein or peptide: TasH
    • RNA: RNA (36-MER)
    • DNA: DNA (38-MER)
    • DNA: DNA (38-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Cryo-EM structure of TasH-tigRNA (5G)-dsDNA complex

SupramoleculeName: Cryo-EM structure of TasH-tigRNA (5G)-dsDNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Salicola phage CGphi29 (virus)

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Macromolecule #1: TasH

MacromoleculeName: TasH / 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 (36-MER)

MacromoleculeName: RNA (36-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Salicola phage CGphi29 (virus)
Molecular weightTheoretical: 11.964239 KDa
SequenceString:
AGUCGUUCCG 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: 11.60645 KDa
SequenceString:
(DC)(DC)(DC)(DT)(DA)(DA)(DG)(DG)(DC)(DA) (DA)(DT)(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: 1 / Classification: DNA
Source (natural)Organism: Salicola phage CGphi29 (virus)
Molecular weightTheoretical: 11.784576 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)(DA)(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: 1 / 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: 2.62 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 314173
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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