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- EMDB-22876: Cryo-EM Structure of the Desulfovibrio vulgaris Type I-C Apo Cascade -

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Basic information

Entry
Database: EMDB / ID: EMD-22876
TitleCryo-EM Structure of the Desulfovibrio vulgaris Type I-C Apo Cascade
Map data
Sample
  • Complex: Type I-C Apo Cascade
    • Protein or peptide: CRISPR-associated protein, CT1134 family
    • 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)
KeywordsCRISPR / Cascade / RNA BINDING PROTEIN-RNA complex
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 (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria) / Desulfovibrio vulgaris str. Hildenborough (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsO'Brien R / Wrapp D / Bravo JPK / Schwartz E / Taylor D
Funding support United States, 2 items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR160088 United States
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis for assembly of non-canonical small subunits into type I-C Cascade.
Authors: Roisin E O'Brien / Inês C Santos / Daniel Wrapp / Jack P K Bravo / Evan A Schwartz / Jennifer S Brodbelt / David W Taylor /
Abstract: Bacteria and archaea employ CRISPR (clustered, regularly, interspaced, short palindromic repeats)-Cas (CRISPR-associated) systems as a type of adaptive immunity to target and degrade foreign nucleic ...Bacteria and archaea employ CRISPR (clustered, regularly, interspaced, short palindromic repeats)-Cas (CRISPR-associated) systems as a type of adaptive immunity to target and degrade foreign nucleic acids. While a myriad of CRISPR-Cas systems have been identified to date, type I-C is one of the most commonly found subtypes in nature. Interestingly, the type I-C system employs a minimal Cascade effector complex, which encodes only three unique subunits in its operon. Here, we present a 3.1 Å resolution cryo-EM structure of the Desulfovibrio vulgaris type I-C Cascade, revealing the molecular mechanisms that underlie RNA-directed complex assembly. We demonstrate how this minimal Cascade utilizes previously overlooked, non-canonical small subunits to stabilize R-loop formation. Furthermore, we describe putative PAM and Cas3 binding sites. These findings provide the structural basis for harnessing the type I-C Cascade as a genome-engineering tool.
History
DepositionOct 20, 2020-
Header (metadata) releaseNov 11, 2020-
Map releaseNov 11, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.21
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.21
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kha
  • Surface level: 1.21
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22876.png

Downloads & links

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Map

FileDownload / File: emd_22876.map.gz / Format: CCP4 / Size: 6.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.047 Å
Density
Contour LevelBy AUTHOR: 1.21 / Movie #1: 1.21
Minimum - Maximum-8.471729 - 14.359439
Average (Standard dev.)0.000000000013258 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions10918888
Spacing18810988
CellA: 196.83601 Å / B: 114.12301 Å / C: 92.136 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z18810988
origin x/y/z0.0000.0000.000
length x/y/z196.836114.12392.136
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS18810988
D min/max/mean-8.47214.3590.000

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Supplemental data

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Half map: #1

Fileemd_22876_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_22876_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 Apo Cascade

EntireName: Type I-C Apo Cascade
Components
  • Complex: Type I-C Apo Cascade
    • Protein or peptide: CRISPR-associated protein, CT1134 family
    • 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)

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Supramolecule #1: Type I-C Apo Cascade

SupramoleculeName: Type I-C Apo Cascade / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Minimal CRISPR Cascade
Source (natural)Organism: Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303
Molecular weightTheoretical: 371 KDa

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Macromolecule #1: CRISPR-associated protein, CT1134 family

MacromoleculeName: CRISPR-associated protein, CT1134 family / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303
Molecular weightTheoretical: 25.250969 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: TYGIRLRVWG DYACFTRPEM KVERVSYDVM PPSAARGILE AIHWKPAIRW IVDRIHVLRP IVFDNVRRNE VSSKIPKPNP ATAMRDRKP LYFLVDDGSN RQQRAATLLR NVDYVIEAHF ELTDKAGAED NAGKHLDIFR RRARAGQSFQ QPCLGCREFP A SFELLEGD ...String:
TYGIRLRVWG DYACFTRPEM KVERVSYDVM PPSAARGILE AIHWKPAIRW IVDRIHVLRP IVFDNVRRNE VSSKIPKPNP ATAMRDRKP LYFLVDDGSN RQQRAATLLR NVDYVIEAHF ELTDKAGAED NAGKHLDIFR RRARAGQSFQ QPCLGCREFP A SFELLEGD VPLSCYAGEK RDLGYMLLDI DFERDMTPLF FKAVMEDGVI TPPSRTSPEV RA

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 (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303
Molecular weightTheoretical: 32.358912 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303
Molecular weightTheoretical: 59.310883 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: WENTSYILGV DAKGKQERTD KCHAAFIAHI KAYCDTADQD LAAVLQFLEH GEKDLSAFPV SEEVIGSNIV FRIEGEPGFV HERPAARQA WANCLNRREQ GLCGQCLITG ERQKPIAQLH PSIKGGRDGV RGAQAVASIV SFNNTAFESY GKEQSINAPV S QEAAFSYV ...String:
WENTSYILGV DAKGKQERTD KCHAAFIAHI KAYCDTADQD LAAVLQFLEH GEKDLSAFPV SEEVIGSNIV FRIEGEPGFV HERPAARQA WANCLNRREQ GLCGQCLITG ERQKPIAQLH PSIKGGRDGV RGAQAVASIV SFNNTAFESY GKEQSINAPV S QEAAFSYV TALNYLLNPS NRQKVTIADA TVVFWAERSS PAEDIFAGMF DPPSTTAKPE SSNGTPPEDS EEGSQPDTAR DD PHAAARM HDLLVAIRSG KRATDIMPDM DESVRFHVLG LSPNAARLSV RFWEVDTVGH MLDKVGRHYR ELEIIPQFNN EQE FPSLST LLRQTAVLNK TENISPVLAG GLFRAMLTGG PYPQSLLPAV LGRIRAEHAR PEDKSRYRLE VVTYYRAALI KAYL IRNRK LEVPVSLDPA RTDRPYLLGR LFAVLEKAQE DAVPGANATI KDRYLASASA NPGQVFHMLL KNASNHTAKL RKDPE RKGS 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 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303
Molecular weightTheoretical: 14.017981 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 #5: RNA (45-MER)

MacromoleculeName: RNA (45-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Desulfovibrio vulgaris str. Hildenborough (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303
Molecular weightTheoretical: 14.457629 KDa
SequenceString:
UGGAUUGAAA CGCCAUGCUC AGGCUGGCGA GUGCGCCACU CAUCA

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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

Concentration0.3 mg/mL
BufferpH: 7.5
GridModel: C-flat-2/2 / Material: COPPER / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5400 / Average electron dose: 33.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 850000
Startup modelType of model: NONE
Final reconstructionNumber classes used: 3 / Resolution.type: BY AUTHOR / Resolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 156524
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 18 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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