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- EMDB-39200: Type I-FHNH Cascade-dsDNA R-loop complex -

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Basic information

Entry
Database: EMDB / ID: EMD-39200
TitleType I-FHNH Cascade-dsDNA R-loop complex
Map data
Sample
  • Complex: Type I-FHNH Cascade-dsDNA R-loop complex
    • Protein or peptide: Cas5f
    • Protein or peptide: Cas8f fusion with HNH
    • Protein or peptide: Cas6f
    • RNA: 60-nt crRNA
    • Other: TS
    • Protein or peptide: Cas7f
    • Other: NTS
Keywordsprotein / RNA / RNA BINDING PROTEIN/DNA/RNA / RNA BINDING PROTEIN-DNA-RNA complex
Biological speciesSelenomonas sp. (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsLi Z
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2024
Title: Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems.
Authors: Chendi Zhang / Fugen Chen / Feng Wang / Haijiang Xu / Jialin Xue / Zhuang Li /
Abstract: The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently ...The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-F and I-E Cascade complexes at different states are reported. In type I-F Cascade, Cas8f loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8f to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-E Cascade, Cas5e domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.
History
DepositionFeb 22, 2024-
Header (metadata) releaseJul 31, 2024-
Map releaseJul 31, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_39200.png

Downloads & links

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Map

FileDownload / File: emd_39200.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.010596851 - 0.027250687
Average (Standard dev.)0.000049116254 (±0.0013593901)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 272.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_39200_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_39200_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 : Type I-FHNH Cascade-dsDNA R-loop complex

EntireName: Type I-FHNH Cascade-dsDNA R-loop complex
Components
  • Complex: Type I-FHNH Cascade-dsDNA R-loop complex
    • Protein or peptide: Cas5f
    • Protein or peptide: Cas8f fusion with HNH
    • Protein or peptide: Cas6f
    • RNA: 60-nt crRNA
    • Other: TS
    • Protein or peptide: Cas7f
    • Other: NTS

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Supramolecule #1: Type I-FHNH Cascade-dsDNA R-loop complex

SupramoleculeName: Type I-FHNH Cascade-dsDNA R-loop complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3, #5-#6, #4, #7
Source (natural)Organism: Selenomonas sp. (bacteria)

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Macromolecule #1: Cas5f

MacromoleculeName: Cas5f / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 28.703135 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 #2: Cas8f fusion with HNH

MacromoleculeName: Cas8f fusion with HNH / 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 BL21(DE3) (bacteria)
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: Cas6f

MacromoleculeName: Cas6f / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 20.735873 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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: Cas7f

MacromoleculeName: Cas7f / type: protein_or_peptide / ID: 4 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 38.700172 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 #5: 60-nt crRNA

MacromoleculeName: 60-nt crRNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 19.440611 KDa
SequenceString:
UUUAGAAGGA GAAGUCAUUU AAUAAGGCCA CUGUUAAAAA GUGUACCGCC GGAUAGGCGG

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Macromolecule #6: TS

MacromoleculeName: TS / type: other / ID: 6 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 15.068426 KDa
SequenceString:
(DG)(DC)(DC)(DA)(DA)(DG)(DC)(DT)(DT)(DT) (DT)(DT)AACAG(DT)GG CC(DT)(DT)A(DT)(DT)AA A(DT)GAC(DT)(DT)C(DT)C CGC(DT)AA(DT)AC
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #7: NTS

MacromoleculeName: NTS / type: other / ID: 7 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 3.541243 KDa
SequenceString:
G(DT)A(DT)(DT)AGCGG A
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 48756
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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