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- EMDB-27402: type I-C Cascade -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-27402
Titletype I-C Cascade
Map data
Sample
  • Complex: type I-C Cascade
    • 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 (48-MER)
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 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/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)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 21, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateMar 15, 2023-
Current statusMar 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27402.png

Downloads & links

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Map

FileDownload / File: emd_27402.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.94 Å
Density
Contour LevelBy AUTHOR: 0.558
Minimum - Maximum-2.278422 - 4.5415106
Average (Standard dev.)-0.00011986117 (±0.057638418)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 413.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_27402_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_27402_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

EntireName: type I-C Cascade
Components
  • Complex: type I-C Cascade
    • 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 (48-MER)

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Supramolecule #1: type I-C Cascade

SupramoleculeName: type I-C Cascade / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Desulfovibrio vulgaris (bacteria) / Strain: Hildenborough

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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

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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

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Macromolecule #3: CRISPR-associated protein, CT1133 family

MacromoleculeName: CRISPR-associated protein, CT1133 family / type: protein_or_peptide / ID: 3 / 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

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Macromolecule #4: CRISPR-associated protein, CT1133 family

MacromoleculeName: CRISPR-associated protein, CT1133 family / type: protein_or_peptide / ID: 4 / 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

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Macromolecule #5: RNA (48-MER)

MacromoleculeName: RNA (48-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Desulfovibrio vulgaris (bacteria) / Strain: Hildenborough
Molecular weightTheoretical: 15.476264 KDa
SequenceString:
GGAUUGAAAC GCCAUGCUCA GGCUGGCGAG UGCGCGCCAC UCAUCAAG

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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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.5 e/Å2

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Image processing

Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 73220

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