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- PDB-7kha: Cryo-EM Structure of the Desulfovibrio vulgaris Type I-C Apo Cascade -

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Basic information

Entry
Database: PDB / ID: 7kha
TitleCryo-EM Structure of the Desulfovibrio vulgaris Type I-C Apo Cascade
Components
  • (CRISPR-associated protein, CT1133 family) x 2
  • CRISPR-associated protein, CT1134 family
  • CRISPR-associated protein, TM1801 family
  • RNA (45-MER)
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / Cascade / 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
RNA / RNA (> 10) / CRISPR-associated protein, TM1801 family / CRISPR-associated protein, CT1133 family / pre-crRNA processing endonuclease
Similarity search - Component
Biological speciesDesulfovibrio vulgaris (bacteria)
Desulfovibrio vulgaris str. Hildenborough (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsO'Brien, R. / Wrapp, D. / Bravo, J.P.K. / Schwartz, E. / Taylor, D.
Funding support United States, 2items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 11, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 14, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: CRISPR-associated protein, CT1134 family
B: CRISPR-associated protein, TM1801 family
C: CRISPR-associated protein, TM1801 family
D: CRISPR-associated protein, TM1801 family
E: CRISPR-associated protein, TM1801 family
F: CRISPR-associated protein, TM1801 family
G: CRISPR-associated protein, TM1801 family
H: CRISPR-associated protein, TM1801 family
I: CRISPR-associated protein, CT1133 family
K: CRISPR-associated protein, CT1133 family
L: CRISPR-associated protein, CT1133 family
J: RNA (45-MER)


Theoretical massNumber of molelcules
Total (without water)353,56812
Polymers353,56812
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CRISPR-associated protein, CT1134 family


Mass: 25250.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303 / Gene: DVUA0130 / Plasmid: D. vulgaris Cascade/I-C (Cas5c-Cas8c-Cas7)/pHMGWA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): NiCo21(DE3) / References: UniProt: Q72WF9
#2: Protein
CRISPR-associated protein, TM1801 family


Mass: 32358.912 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303 / Gene: DVUA0132 / Plasmid: D. vulgaris Cascade/I-C (Cas5c-Cas8c-Cas7)/pHMGWA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): NiCo21(DE3) / References: UniProt: Q72WF7
#3: Protein CRISPR-associated protein, CT1133 family


Mass: 59310.883 Da / Num. of mol.: 1 / Fragment: UNP residues 80-612
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303 / Gene: DVUA0131 / Plasmid: D. vulgaris Cascade/I-C (Cas5c-Cas8c-Cas7)/pHMGWA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): NiCo21(DE3) / References: UniProt: Q72WF8
#4: Protein CRISPR-associated protein, CT1133 family


Mass: 14017.981 Da / Num. of mol.: 2 / Fragment: Cas8c C-terminal domain (UNP residues 489-612)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303 / Gene: DVUA0131 / Plasmid: D. vulgaris Cascade/I-C (Cas5c-Cas8c-Cas7)/pHMGWA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): NiCo21(DE3) / References: UniProt: Q72WF8
#5: RNA chain RNA (45-MER)


Mass: 14457.629 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfovibrio vulgaris str. Hildenborough (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303 / Plasmid: D. vulgaris sp2 CRISPR/pACYCDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): NiCo21(DE3)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Type I-C Apo Cascade / Type: COMPLEX / Details: Minimal CRISPR Cascade / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.371 MDa / Experimental value: YES
Source (natural)Organism: Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303) (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: NiCo21(DE3) / Plasmid: D. vulgaris Cascade/I-C (Cas5c-Cas8c-Cas7)/pHMGWA
Buffer solutionpH: 7.5
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 33 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5400

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameCategory
2Leginonimage acquisition
4WarpCTF correction
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
14ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 850000
3D reconstructionResolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 156524 / Num. of class averages: 3 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00723973
ELECTRON MICROSCOPYf_angle_d0.70832628
ELECTRON MICROSCOPYf_dihedral_angle_d15.37314526
ELECTRON MICROSCOPYf_chiral_restr0.0493626
ELECTRON MICROSCOPYf_plane_restr0.0054128

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