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- PDB-6lnb: CryoEM structure of Cascade-TniQ-dsDNA complex -

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Basic information

Entry
Database: PDB / ID: 6lnb
TitleCryoEM structure of Cascade-TniQ-dsDNA complex
Components
  • (CRISPR-associated protein ...) x 3
  • CRISPR RNA (60-MER)
  • Non-target DNA strand (51-MER)
  • Target DNA strand (51-MER)
  • Transposition protein TniQ
KeywordsIMMUNE SYSTEM / Type I-F CRISPR-Cas system / Csy Cascade / Tn7-like transposons / RNA-guided tranposition / transposase subunit
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesVibrio cholerae (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsWang, B. / Xu, W. / Yang, H.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971135 China
CitationJournal: Cell Res / Year: 2020
Title: Structural basis of a Tn7-like transposase recruitment and DNA loading to CRISPR-Cas surveillance complex.
Authors: Beibei Wang / Wenhao Xu / Hui Yang /
History
DepositionDec 28, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 19, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 27, 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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Structure viewerMolecule:
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Assembly

Deposited unit
G: CRISPR-associated protein Cas7
F: CRISPR-associated protein Cas7
E: CRISPR-associated protein Cas7
D: CRISPR-associated protein Cas7
C: CRISPR-associated protein Cas7
B: CRISPR-associated protein Cas7
H: CRISPR-associated protein Cas8
A: CRISPR-associated protein Cas6
J: Transposition protein TniQ
M: CRISPR RNA (60-MER)
N: Target DNA strand (51-MER)
O: Non-target DNA strand (51-MER)
I: Transposition protein TniQ


Theoretical massNumber of molelcules
Total (without water)477,46813
Polymers477,46813
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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CRISPR-associated protein ... , 3 types, 8 molecules GFEDCBHA

#1: Protein
CRISPR-associated protein Cas7


Mass: 40030.156 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Plasmid: pRSFDuet-SUMO / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
#2: Protein CRISPR-associated protein Cas8


Mass: 72294.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Plasmid: pCDFDuet / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
#3: Protein CRISPR-associated protein Cas6


Mass: 23098.365 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Plasmid: pETDuet / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)

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DNA chain , 2 types, 2 molecules NO

#6: DNA chain Target DNA strand (51-MER)


Mass: 15856.158 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#7: DNA chain Non-target DNA strand (51-MER)


Mass: 15535.982 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Protein / RNA chain , 2 types, 3 molecules JIM

#4: Protein Transposition protein TniQ


Mass: 45597.867 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Plasmid: pRSFDuet-SUMO / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
#5: RNA chain CRISPR RNA (60-MER)


Mass: 19305.428 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Plasmid: pETDuet / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(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: CryoEM structure of Cascade-TniQ-dsDNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Vibrio cholerae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
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 FIELDBright-field microscopy
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 48 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 206913 / Symmetry type: POINT

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