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- EMDB-27409: type I-C Cascade bound to AcrIC4 -

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Basic information

Entry
Database: EMDB / ID: EMD-27409
Titletype I-C Cascade bound to AcrIC4
Map data
Sample
  • Complex: type I-C Cascade bound to AcrIC4
    • Protein or peptide: pre-crRNA processing endonuclease
    • Protein or peptide: CRISPR-associated protein, TM1801 family
    • Protein or peptide: CRISPR-associated protein, CT1133 family
    • Protein or peptide: CRISPR-associated protein, CT1133 family
    • RNA: RNA (45-MER)
    • Protein or peptide: AcrIC4
KeywordsCRISPR / type I-C / anti-CRISPR / Cascade / type I-C Cascade / AcrIC4 / DNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding
Similarity search - Function
CRISPR-associated protein Csd2 / CRISPR-associated protein Cas7, subtype I-B/I-C / CRISPR-associated protein Cas7 / CRISPR-associated protein, Csd1-type / CRISPR-associated protein (Cas_Csd1) / CRISPR pre-crRNA endoribonuclease Cas5d / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
CRISPR-associated protein, TM1801 family / CRISPR-associated protein, CT1133 family / pre-crRNA processing endonuclease
Similarity search - Component
Biological speciesDesulfovibrio vulgaris (bacteria) / Pseudomonas aeruginosa phage DMS3mVir (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsO'Brien RE / Bravo JPK / Ramos D / Hibshman GN / Wright JT / Taylor DW
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR160088 United States
CitationJournal: Mol Cell / Year: 2023
Title: Structural snapshots of R-loop formation by a type I-C CRISPR Cascade.
Authors: Roisin E O'Brien / Jack P K Bravo / Delisa Ramos / Grace N Hibshman / Jacquelyn T Wright / David W Taylor /
Abstract: Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. Here, we present structures of D. vulgaris type I-C Cascade at various ...Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. Here, we present structures of D. vulgaris type I-C Cascade at various stages of double-stranded (ds)DNA target capture, revealing mechanisms that underpin PAM recognition and Cascade allosteric activation. We uncover an interesting mechanism of non-target strand (NTS) DNA stabilization via stacking interactions with the "belly" subunits, securing the NTS in place. This "molecular seatbelt" mechanism facilitates efficient R-loop formation and prevents dsDNA reannealing. Additionally, we provide structural insights into how two anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking PAM scanning. These observations form a structural basis for directional R-loop formation and reveal how different Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.
History
DepositionJun 22, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27409.png

Downloads & links

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Map

FileDownload / File: emd_27409.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 420 pix.
= 394.8 Å		0.94 Å/pix.
x 420 pix.
= 394.8 Å		0.94 Å/pix.
x 420 pix.
= 394.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.94 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.253
Minimum - Maximum-0.9421694 - 1.674114
Average (Standard dev.)-0.00014695861 (±0.04464671)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 394.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27409_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27409_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-C Cascade bound to AcrIC4

EntireName: type I-C Cascade bound to AcrIC4
Components
  • Complex: type I-C Cascade bound to AcrIC4
    • Protein or peptide: pre-crRNA processing endonuclease
    • Protein or peptide: CRISPR-associated protein, TM1801 family
    • Protein or peptide: CRISPR-associated protein, CT1133 family
    • Protein or peptide: CRISPR-associated protein, CT1133 family
    • RNA: RNA (45-MER)
    • Protein or peptide: AcrIC4

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Supramolecule #1: type I-C Cascade bound to AcrIC4

SupramoleculeName: type I-C Cascade bound to AcrIC4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6

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Macromolecule #1: pre-crRNA processing endonuclease

MacromoleculeName: pre-crRNA processing endonuclease / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Desulfovibrio vulgaris (bacteria)
Strain: ATCC 29579 / DSM 644 / NCIMB 8303 / VKM B-1760 / Hildenborough
Molecular weightTheoretical: 25.977857 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTHGAVKTYG IRLRVWGDYA CFTRPEMKVE RVSYDVMPPS AARGILEAIH WKPAIRWIVD RIHVLRPIVF DNVRRNEVSS KIPKPNPAT AMRDRKPLYF LVDDGSNRQQ RAATLLRNVD YVIEAHFELT DKAGAEDNAG KHLDIFRRRA RAGQSFQQPC L GCREFPAS ...String:
MTHGAVKTYG IRLRVWGDYA CFTRPEMKVE RVSYDVMPPS AARGILEAIH WKPAIRWIVD RIHVLRPIVF DNVRRNEVSS KIPKPNPAT AMRDRKPLYF LVDDGSNRQQ RAATLLRNVD YVIEAHFELT DKAGAEDNAG KHLDIFRRRA RAGQSFQQPC L GCREFPAS FELLEGDVPL SCYAGEKRDL GYMLLDIDFE RDMTPLFFKA VMEDGVITPP SRTSPEVRA

UniProtKB: pre-crRNA processing endonuclease

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Macromolecule #2: CRISPR-associated protein, TM1801 family

MacromoleculeName: CRISPR-associated protein, TM1801 family / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Desulfovibrio vulgaris (bacteria)
Strain: ATCC 29579 / DSM 644 / NCIMB 8303 / VKM B-1760 / Hildenborough
Molecular weightTheoretical: 32.358912 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTAIANRYEF VLLFDVENGN PNGDPDAGNM PRIDPETGHG LVTDVCLKRK IRNHVALTKE GAERFNIYIQ EKAILNETHE RAYTACDLK PEPKKLPKKV EDAKRVTDWM CTNFYDIRTF GAVMTTEVNC GQVRGPVQMA FARSVEPVVP QEVSITRMAV T TKAEAEKQ ...String:
MTAIANRYEF VLLFDVENGN PNGDPDAGNM PRIDPETGHG LVTDVCLKRK IRNHVALTKE GAERFNIYIQ EKAILNETHE RAYTACDLK PEPKKLPKKV EDAKRVTDWM CTNFYDIRTF GAVMTTEVNC GQVRGPVQMA FARSVEPVVP QEVSITRMAV T TKAEAEKQ QGDNRTMGRK HIVPYGLYVA HGFISAPLAE KTGFSDEDLT LFWDALVNMF EHDRSAARGL MSSRKLIVFK HQ NRLGNAP AHKLFDLVKV SRAEGSSGPA RSFADYAVTV GQAPEGVEVK EML

UniProtKB: CRISPR-associated protein, TM1801 family

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Macromolecule #3: CRISPR-associated protein, CT1133 family

MacromoleculeName: CRISPR-associated protein, CT1133 family / type: protein_or_peptide / ID: 3 / Details: Cas8c / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfovibrio vulgaris (bacteria)
Strain: ATCC 29579 / DSM 644 / NCIMB 8303 / VKM B-1760 / Hildenborough
Molecular weightTheoretical: 68.123219 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MILQALHGYY QRMSADPDAG MPPYGTSMEN ISFALVLDAK GTLRGIEDLR EQEGKKLRPR KMLVPIAEKK GNGIKPNFLW ENTSYILGV DAKGKQERTD KCHAAFIAHI KAYCDTADQD LAAVLQFLEH GEKDLSAFPV SEEVIGSNIV FRIEGEPGFV H ERPAARQA ...String:
MILQALHGYY QRMSADPDAG MPPYGTSMEN ISFALVLDAK GTLRGIEDLR EQEGKKLRPR KMLVPIAEKK GNGIKPNFLW ENTSYILGV DAKGKQERTD KCHAAFIAHI KAYCDTADQD LAAVLQFLEH GEKDLSAFPV SEEVIGSNIV FRIEGEPGFV H ERPAARQA WANCLNRREQ GLCGQCLITG ERQKPIAQLH PSIKGGRDGV RGAQAVASIV SFNNTAFESY GKEQSINAPV SQ EAAFSYV TALNYLLNPS NRQKVTIADA TVVFWAERSS PAEDIFAGMF DPPSTTAKPE SSNGTPPEDS EEGSQPDTAR DDP HAAARM HDLLVAIRSG KRATDIMPDM DESVRFHVLG LSPNAARLSV RFWEVDTVGH MLDKVGRHYR ELEIIPQFNN EQEF PSLST LLRQTAVLNK TENISPVLAG GLFRAMLTGG PYPQSLLPAV LGRIRAEHAR PEDKSRYRLE VVTYYRAALI KAYLI RNRK LEVPVSLDPA RTDRPYLLGR LFAVLEKAQE DAVPGANATI KDRYLASASA NPGQVFHMLL KNASNHTAKL RKDPER KGS AIHYEIMMQE IIDNISDFPV TMSSDEQGLF MIGYYHQRKA LFTKKNKEN

UniProtKB: CRISPR-associated protein, CT1133 family

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Macromolecule #4: CRISPR-associated protein, CT1133 family

MacromoleculeName: CRISPR-associated protein, CT1133 family / type: protein_or_peptide / ID: 4 / Details: Cas11c / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Desulfovibrio vulgaris (bacteria)
Strain: ATCC 29579 / DSM 644 / NCIMB 8303 / VKM B-1760 / Hildenborough
Molecular weightTheoretical: 14.017981 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
VSLDPARTDR PYLLGRLFAV LEKAQEDAVP GANATIKDRY LASASANPGQ VFHMLLKNAS NHTAKLRKDP ERKGSAIHYE IMMQEIIDN ISDFPVTMSS DEQGLFMIGY YHQRKALFTK KNKEN

UniProtKB: CRISPR-associated protein, CT1133 family

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Macromolecule #6: AcrIC4

MacromoleculeName: AcrIC4 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa phage DMS3mVir (virus)
Molecular weightTheoretical: 6.659252 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MDNKITPADE EKIREWLNCE EASVDNDGDV WVAVPMTGHW LSDEQKAKYI EWRGDET

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Macromolecule #5: RNA (45-MER)

MacromoleculeName: RNA (45-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Desulfovibrio vulgaris (bacteria) / Strain: Hildenborough
Molecular weightTheoretical: 14.472646 KDa
SequenceString:
GGAUUGAAAC GCCAUGCUCA GGCUGGCGAG UGCGCGCCAC UCAUC

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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 GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.5 e/Å2
Electron beamAcceleration voltage: 200 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

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 21651
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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