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- PDB-6pig: V. cholerae TniQ-Cascade complex, closed conformation -

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Basic information

Entry
Database: PDB / ID: 6pig
TitleV. cholerae TniQ-Cascade complex, closed conformation
Components
  • (TniQ monomer ...) x 2
  • RNA (60-MER)
  • cas5_8 naturally occurring fusion protein from Vibrio cholerae transposon Tn6677
  • cas7 type I-F CRISPR-associated protein Csy3
  • type I-F CRISPR-associated endoribonuclease Cas6/Csy4
KeywordsRNA BINDING PROTEIN/RNA / CRISPR/Cas / Cascade / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesVibrio cholerae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHalpin-Healy, T. / Klompe, S. / Sternberg, S.H.
CitationJournal: Nature / Year: 2020
Title: Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system.
Authors: Tyler S Halpin-Healy / Sanne E Klompe / Samuel H Sternberg / Israel S Fernández /
Abstract: Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements. Type I CRISPR-Cas systems typically ...Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.
History
DepositionJun 26, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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
Revision 1.2Jun 24, 2020Group: Structure summary / Category: audit_author / struct / Item: _audit_author.name / _struct.title
Revision 1.3Mar 20, 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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Structure visualization

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Assembly

Deposited unit
1: RNA (60-MER)
A: cas7 type I-F CRISPR-associated protein Csy3
B: cas7 type I-F CRISPR-associated protein Csy3
C: cas7 type I-F CRISPR-associated protein Csy3
D: cas7 type I-F CRISPR-associated protein Csy3
E: cas7 type I-F CRISPR-associated protein Csy3
F: cas7 type I-F CRISPR-associated protein Csy3
G: cas5_8 naturally occurring fusion protein from Vibrio cholerae transposon Tn6677
H: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
I: TniQ monomer 1
J: TniQ monomer 2


Theoretical massNumber of molelcules
Total (without water)417,18111
Polymers417,18111
Non-polymers00
Water0
1


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

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Components

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RNA chain , 1 types, 1 molecules 1

#1: RNA chain RNA (60-MER)


Mass: 19237.338 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vibrio cholerae (bacteria)

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Protein , 3 types, 8 molecules ABCDEFGH

#2: Protein
cas7 type I-F CRISPR-associated protein Csy3


Mass: 39020.035 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli)
#3: Protein cas5_8 naturally occurring fusion protein from Vibrio cholerae transposon Tn6677


Mass: 57064.711 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli)
#4: Protein type I-F CRISPR-associated endoribonuclease Cas6/Csy4


Mass: 22926.184 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli)

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TniQ monomer ... , 2 types, 2 molecules IJ

#5: Protein TniQ monomer 1


Mass: 41517.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli)
#6: Protein TniQ monomer 2


Mass: 42315.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli)

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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: VC-Tn667 transposon encoded CRISPR/Cas effector / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)Organism: Vibrio cholerae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMhepes-kohhepes1
2200 mMsodium chlorideNaClSodium chloride1
32 mMditiotreitolDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0238 / Classification: refinement
EM softwareName: RELION / Version: 3 / Category: final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87000 / Symmetry type: POINT

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