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- PDB-6vbw: Cryo-EM structure of Cascade-TniQ-dsDNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 6vbw
TitleCryo-EM structure of Cascade-TniQ-dsDNA ternary complex
Components
  • Cas6
  • Cas7
  • Cas8
  • DNA (100-MER)
  • DNA (5'-D(*GP*CP*AP*GP*TP*CP*AP*TP*CP*AP*CP*CP*AP*AP*TP*TP*TP*AP*TP*TP*TP*A)-3')
  • RNA (61-MER)
  • TniQ
KeywordsIMMUNE SYSTEM / CRISPR-Cas system / Cascade- TniQ
Function / homologyCRISPR-associated endoribonuclease Cas6/Csy4 / Alpha-Beta Plaits / 2-Layer Sandwich / Alpha Beta / DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10)
Function and homology information
Biological speciesVibrio cholerae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsJia, N. / Patel, D.J.
CitationJournal: Cell Res / Year: 2020
Title: Structure-function insights into the initial step of DNA integration by a CRISPR-Cas-Transposon complex.
Authors: Ning Jia / Wei Xie / M Jason de la Cruz / Edward T Eng / Dinshaw J Patel /
History
DepositionDec 19, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 29, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
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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Structure visualization

Movie
  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-21146
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
M: DNA (5'-D(*GP*CP*AP*GP*TP*CP*AP*TP*CP*AP*CP*CP*AP*AP*TP*TP*TP*AP*TP*TP*TP*A)-3')
L: DNA (100-MER)
K: RNA (61-MER)
A: Cas8
B: Cas7
C: Cas7
D: Cas7
F: Cas7
E: Cas7
G: Cas7
H: Cas6
I: TniQ
J: TniQ
hetero molecules


Theoretical massNumber of molelcules
Total (without water)483,35317
Polymers483,09113
Non-polymers2624
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area64300 Å2
ΔGint-353 kcal/mol
Surface area143870 Å2

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Components

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DNA chain , 2 types, 2 molecules ML

#1: DNA chain DNA (5'-D(*GP*CP*AP*GP*TP*CP*AP*TP*CP*AP*CP*CP*AP*AP*TP*TP*TP*AP*TP*TP*TP*A)-3')


Mass: 6685.354 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vibrio cholerae (bacteria)
#2: DNA chain DNA (100-MER)


Mass: 30901.777 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vibrio cholerae (bacteria)

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Protein , 4 types, 10 molecules ABCDFEGHIJ

#4: Protein Cas8


Mass: 72294.930 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)
#5: Protein
Cas7


Mass: 39886.031 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)
#6: Protein Cas6


Mass: 23130.430 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)
#7: Protein TniQ


Mass: 45597.867 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli)

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RNA chain / Non-polymers , 2 types, 5 molecules K

#3: RNA chain RNA (61-MER)


Mass: 19566.543 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vibrio cholerae (bacteria)
#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Cascade-TniQ-dsDNA ternary complexCOMPLEX#1-#70RECOMBINANT
2DNA/RNACOMPLEX#1-#2, #61RECOMBINANT
3Cascade ternary complexCOMPLEX#3-#5, #71RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Vibrio cholerae (bacteria)666
23Vibrio cholerae (bacteria)666
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12synthetic construct (others)32630
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.5 / Details: 20 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM DTT
Buffer componentFormula: Tris
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 FIELD
Image recordingElectron dose: 2.16 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: RELION / Version: 2.1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55900 / Symmetry type: POINT

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