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- EMDB-60017: Cryo-EM structure of Cas8-HNH system at target free state -

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Basic information

Entry
Database: EMDB / ID: EMD-60017
TitleCryo-EM structure of Cas8-HNH system at target free state
Map data
Sample
  • Complex: a protein
    • Protein or peptide: a protein
    • Protein or peptide: a protein
    • Protein or peptide: a protein
    • Protein or peptide: a protein
    • RNA: RNA (58-MER)
Keywordsa protein complex / ANTIVIRAL PROTEIN
Biological speciesSelenomonas sp. (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsZhang H / Zhu H / Li X / Liu Y
Funding support China, 1 items
OrganizationGrant numberCountry
Other government China
CitationJournal: EMBO J / Year: 2024
Title: Structural basis for the type I-F Cas8-HNH system.
Authors: Xuzichao Li / Yanan Liu / Jie Han / Lingling Zhang / Zhikun Liu / Lin Wang / Shuqin Zhang / Qian Zhang / Pengyu Fu / Hang Yin / Hongtao Zhu / Heng Zhang /
Abstract: The Cas3 nuclease is utilized by canonical type I CRISPR-Cas systems for processive target DNA degradation, while a newly identified type I-F CRISPR variant employs an HNH nuclease domain from the ...The Cas3 nuclease is utilized by canonical type I CRISPR-Cas systems for processive target DNA degradation, while a newly identified type I-F CRISPR variant employs an HNH nuclease domain from the natural fusion Cas8-HNH protein for precise target cleavage both in vitro and in human cells. Here, we report multiple cryo-electron microscopy structures of the type I-F Cas8-HNH system at different functional states. The Cas8-HNH Cascade complex adopts an overall G-shaped architecture, with the HNH domain occupying the C-terminal helical bundle domain (HB) of the Cas8 protein in canonical type I systems. The Linker region connecting Cas8-NTD and HNH domains adopts a rigid conformation and interacts with the Cas7.6 subunit, enabling the HNH domain to be in a functional position. The full R-loop formation displaces the HNH domain away from the Cas6 subunit, thus activating the target DNA cleavage. Importantly, our results demonstrate that precise target cleavage is dictated by a C-terminal helix of the HNH domain. Together, our work not only delineates the structural basis for target recognition and activation of the type I-F Cas8-HNH system, but also guides further developments leveraging this system for precise DNA editing.
History
DepositionMay 3, 2024-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60017.png

Downloads & links

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Map

FileDownload / File: emd_60017.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 360 pix.
= 308.16 Å		0.86 Å/pix.
x 360 pix.
= 308.16 Å		0.86 Å/pix.
x 360 pix.
= 308.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.856 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.052286394 - 2.6728516
Average (Standard dev.)0.0022343453 (±0.034469627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 308.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_60017_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_60017_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : a protein

EntireName: a protein
Components
  • Complex: a protein
    • Protein or peptide: a protein
    • Protein or peptide: a protein
    • Protein or peptide: a protein
    • Protein or peptide: a protein
    • RNA: RNA (58-MER)

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Supramolecule #1: a protein

SupramoleculeName: a protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Selenomonas sp. (bacteria)

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Macromolecule #1: a protein

MacromoleculeName: a protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 38.700172 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAANKKATNV TLKSRPENLS FARCLNTTEA KFWQTDFLKR HTFKLPLLIT DKAVLASKGH EMPPDKLEKE IMDPNPQKSQ SCTLSTECD TLRIDFGIKV LPVKESMYSC SDYNYRTAIY QKIDEYIAED GFLTLAKRYV NNIANARFLW RNRKGAEIIE T IVTIEDKE ...String:
MAANKKATNV TLKSRPENLS FARCLNTTEA KFWQTDFLKR HTFKLPLLIT DKAVLASKGH EMPPDKLEKE IMDPNPQKSQ SCTLSTECD TLRIDFGIKV LPVKESMYSC SDYNYRTAIY QKIDEYIAED GFLTLAKRYV NNIANARFLW RNRKGAEIIE T IVTIEDKE YPSFNSKSFN LDTFVEDNAT INEIAQQIAD TFAGKREYLN IYVTCFVKIG CAMEVYPSQE MTFDDDDKGK KL FKFEGSA GMHSQKINNA LRTIDTWYPD YTTYEFPIPV ENYGAARSIG IPFRPDTKSF YKLIDRMILK NEDLPIEDKH YVM AILIRG GMFSKKQEK

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Macromolecule #2: a protein

MacromoleculeName: a protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 39.339742 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLRNKILAAI SQKIPEEQKI NKYIEGLFQS IDKNHLATHV AKFTETNSPG NIGAYDILSS DMNCGYLDTA NAGWKEPDIV TNDAKYKRP QGFVAMEMSD GRTVMEHLQE DSAELRHEME ELTDKYDEIR DGILNMPSMQ PYRTNQFIKQ VFFPVGGSYH L LSILPSTV ...String:
MLRNKILAAI SQKIPEEQKI NKYIEGLFQS IDKNHLATHV AKFTETNSPG NIGAYDILSS DMNCGYLDTA NAGWKEPDIV TNDAKYKRP QGFVAMEMSD GRTVMEHLQE DSAELRHEME ELTDKYDEIR DGILNMPSMQ PYRTNQFIKQ VFFPVGGSYH L LSILPSTV LNYEVSDRLY RSKIPKIRLR LLSSNAASTT GSRLVSKNKW PLVFQALPPK FLEKNLAKAL DKEYLLPDIN ID ELEGVDN GCLIDEALLP LIIDEGKRKG EGNYRPRHLR DERKEETVQA FLDKYGYCNI PVGYEVHHIV PLSQGGADSI KNM IMLSIE HHERVTEAHA SYFKWRNT

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Macromolecule #3: a protein

MacromoleculeName: a protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 28.703135 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MMKGYILLEK VNIENANAFN NIIVGIPAIT SFLGFARALE RKLNAKEIAI RINGVGLEFH EYELKGYKNK RGQYVTSCPL PGSIPGQNE KKLDAHIMNQ AYIDLNMSFL LEVEGPHVDM STCKSIKSTM ETLRIAGGII RNYKKIRLID TLADIPYGYF L TLRQDNLN ...String:
MMKGYILLEK VNIENANAFN NIIVGIPAIT SFLGFARALE RKLNAKEIAI RINGVGLEFH EYELKGYKNK RGQYVTSCPL PGSIPGQNE KKLDAHIMNQ AYIDLNMSFL LEVEGPHVDM STCKSIKSTM ETLRIAGGII RNYKKIRLID TLADIPYGYF L TLRQDNLN DAAGDDMLDK MIHALQQEDT LVPIAVGFKA LSEVGHVEGQ RDPEKDHCFV ESIFSLGGFE CSKILEDINS CL WRYKTEE GLYLCTII

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Macromolecule #4: a protein

MacromoleculeName: a protein / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 20.735873 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MFSQILIIKP GTGISPNIII SEDIFPVLHS LFVEHDKKFG ITFPAYSFDK KGHLGNIIEV LSEDKEALAS LCLEEHLAEV TDYVKVKKE ITFTDDYVLF KRIREENQYE TTARRMRKRG HTELGRPLEM HIKKKNQQIF CHAYIKVKSA STGQSYNIFL A PTDIKHGS FSAYGLLRGD THA

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Macromolecule #5: RNA (58-MER)

MacromoleculeName: RNA (58-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 22.398293 KDa
SequenceString:
GUUUAGAAGG AUUGCCGUCA GGAAAUUAGG UGCGCUUAGC AGUGUACCGC CGGAUAGGCG GUUUAGAAG

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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

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 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

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 220116
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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