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- EMDB-39706: Cryo-EM structure of Cas8-HNH system at full R-loop state -

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Basic information

Entry
Database: EMDB / ID: EMD-39706
TitleCryo-EM structure of Cas8-HNH system at full R-loop state
Map data
Sample
  • Complex: Cryo-EM structure of Cas8-HNH system at full R-loop state
    • Protein or peptide: type I-F CRISPR-associated protein Csy3
    • Protein or peptide: hypothetical protein J6N51_11000
    • Protein or peptide: HNH endonuclease
    • DNA: DNA (37-MER)
    • DNA: DNA (5'-D(P*GP*TP*GP*CP*GP*GP*A)-3')
    • RNA: RNA (69-MER)
    • Protein or peptide: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
Keywordsa protein complex / ANTIVIRAL PROTEIN/DNA/RNA / ANTIVIRAL PROTEIN-DNA-RNA complex
Biological speciesSelenomonas sp. (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.51 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
DepositionApr 9, 2024-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_39706.png

Downloads & links

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Map

FileDownload / File: emd_39706.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
0.75 Å/pix.
x 360 pix.
= 270. Å		0.75 Å/pix.
x 360 pix.
= 270. Å		0.75 Å/pix.
x 360 pix.
= 270. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.75 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.0016976122 - 1.9552153
Average (Standard dev.)0.0027507138 (±0.038032383)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_39706_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_39706_half_map_2.map
Projections & Slices
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Sample components

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Entire : Cryo-EM structure of Cas8-HNH system at full R-loop state

EntireName: Cryo-EM structure of Cas8-HNH system at full R-loop state
Components
  • Complex: Cryo-EM structure of Cas8-HNH system at full R-loop state
    • Protein or peptide: type I-F CRISPR-associated protein Csy3
    • Protein or peptide: hypothetical protein J6N51_11000
    • Protein or peptide: HNH endonuclease
    • DNA: DNA (37-MER)
    • DNA: DNA (5'-D(P*GP*TP*GP*CP*GP*GP*A)-3')
    • RNA: RNA (69-MER)
    • Protein or peptide: type I-F CRISPR-associated endoribonuclease Cas6/Csy4

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Supramolecule #1: Cryo-EM structure of Cas8-HNH system at full R-loop state

SupramoleculeName: Cryo-EM structure of Cas8-HNH system at full R-loop state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Selenomonas sp. (bacteria)

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Macromolecule #1: type I-F CRISPR-associated protein Csy3

MacromoleculeName: type I-F CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 1
Details: Molecular name from NCBI link:https://www.ncbi.nlm.nih.gov/protein/MBO6292766.1
Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 37.668945 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TLKSRPENLS FARCLNTTEA KFWQTDFLKR HTFKLPLLIT DKAVLASKGH EMPPDKLEKE IMDPNPQKSQ SCTLSTECDT LRIDFGIKV LPVKESMYSC SDYNYRTAIY QKIDEYIAED GFLTLAKRYV NNIANARFLW RNRKGAEIIE TIVTIEDKEY P SFNSKSFN ...String:
TLKSRPENLS FARCLNTTEA KFWQTDFLKR HTFKLPLLIT DKAVLASKGH EMPPDKLEKE IMDPNPQKSQ SCTLSTECDT LRIDFGIKV LPVKESMYSC SDYNYRTAIY QKIDEYIAED GFLTLAKRYV NNIANARFLW RNRKGAEIIE TIVTIEDKEY P SFNSKSFN LDTFVEDNAT INEIAQQIAD TFAGKREYLN IYVTCFVKIG CAMEVYPSQE MTFDDDDKGK KLFKFEGSAG MH SQKINNA LRTIDTWYPD YTTYEFPIPV ENYGAARSIG IPFRPDTKSF YKLIDRMILK NEDLPIEDKH YVMAILIRGG MFS KKQEK

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Macromolecule #2: hypothetical protein J6N51_11000

MacromoleculeName: hypothetical protein J6N51_11000 / type: protein_or_peptide / ID: 2
Details: Molecular name from NCBI link:https://www.ncbi.nlm.nih.gov/protein/MBO6292763.1
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 #3: HNH endonuclease

MacromoleculeName: HNH endonuclease / type: protein_or_peptide / ID: 3
Details: Molecular name from NCBI link:https://www.ncbi.nlm.nih.gov/protein/MBO6292764.1
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 #7: type I-F CRISPR-associated endoribonuclease Cas6/Csy4

MacromoleculeName: type I-F CRISPR-associated endoribonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 7
Details: Molecular name from NCBI link:https://www.ncbi.nlm.nih.gov/protein/MBO6292765.1
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 #4: DNA (37-MER)

MacromoleculeName: DNA (37-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 11.27125 KDa
SequenceString:
(DT)(DG)(DC)(DT)(DA)(DA)(DG)(DC)(DG)(DC) (DA)(DC)(DC)(DT)(DA)(DA)(DT)(DT)(DT)(DC) (DC)(DT)(DG)(DA)(DC)(DG)(DG)(DC)(DA) (DA)(DT)(DC)(DC)(DG)(DC)(DA)(DC)

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Macromolecule #5: DNA (5'-D(P*GP*TP*GP*CP*GP*GP*A)-3')

MacromoleculeName: DNA (5'-D(P*GP*TP*GP*CP*GP*GP*A)-3') / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 2.178447 KDa
SequenceString:
(DG)(DT)(DG)(DC)(DG)(DG)(DA)

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Macromolecule #6: RNA (69-MER)

MacromoleculeName: RNA (69-MER) / type: rna / ID: 6 / 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: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 173047
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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