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- PDB-7u5d: I-F3b Cascade-TniQ full R-loop complex -

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Basic information

Entry
Database: PDB / ID: 7u5d
TitleI-F3b Cascade-TniQ full R-loop complex
Components
  • Cas6
  • Cas7
  • Cas8/5
  • Non-target strand DNA
  • Target strand DNA
  • TniQ
  • crRNA
KeywordsDNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas / Transposon / CAST / Cascade / I-F / I-F3 / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex
Function / homologyDNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10)
Function and homology information
Biological speciesAeromonas salmonicida (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsPark, J.U. / Mehrotra, E. / Kellogg, E.H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00-GM124463 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM144566 United States
CitationJournal: Mol Cell / Year: 2023
Title: Multiple adaptations underly co-option of a CRISPR surveillance complex for RNA-guided DNA transposition.
Authors: Jung-Un Park / Michael T Petassi / Shan-Chi Hsieh / Eshan Mehrotra / Gabriel Schuler / Jagat Budhathoki / Vinh H Truong / Summer B Thyme / Ailong Ke / Elizabeth H Kellogg / Joseph E Peters /
Abstract: CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA- ...CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets. To investigate adaptations allowing CAST elements to utilize attachment sites inaccessible to CRISPR-Cas surveillance complexes, we compared and contrasted PAM sequence requirements in both I-F3b CAST and I-F1 CRISPR-Cas systems. We identify specific amino acids that enable a wider range of PAM sequences to be accommodated in I-F3b CAST elements compared with I-F1 CRISPR-Cas, enabling CAST elements to access attachment sites as sequences drift and evade host surveillance. Together, this evidence points to the central role of TniQ in facilitating the acquisition of CRISPR effector complexes for RNA-guided DNA transposition.
History
DepositionMar 2, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: crRNA
2: Target strand DNA
3: Non-target strand DNA
A: Cas8/5
B: Cas7
C: Cas7
D: Cas7
E: Cas7
F: Cas7
G: Cas7
H: Cas6
I: TniQ
J: TniQ


Theoretical massNumber of molelcules
Total (without water)520,48513
Polymers520,48513
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RNA chain , 1 types, 1 molecules 1

#1: RNA chain crRNA


Mass: 19399.604 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aeromonas salmonicida (bacteria) / Production host: Escherichia coli (E. coli)

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DNA chain , 2 types, 2 molecules 23

#2: DNA chain Target strand DNA


Mass: 35613.680 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Aeromonas salmonicida (bacteria)
#3: DNA chain Non-target strand DNA


Mass: 35737.723 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Aeromonas salmonicida (bacteria)

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Protein , 4 types, 10 molecules ABCDEFGHIJ

#4: Protein Cas8/5


Mass: 78728.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aeromonas salmonicida (bacteria) / Production host: Escherichia coli (E. coli)
#5: Protein
Cas7


Mass: 39105.172 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aeromonas salmonicida (bacteria) / Production host: Escherichia coli (E. coli)
#6: Protein Cas6


Mass: 23640.832 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aeromonas salmonicida (bacteria) / Production host: Escherichia coli (E. coli)
#7: Protein TniQ


Mass: 46366.637 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aeromonas salmonicida (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: I-F3b Cascade-TniQ Full R-loop complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.45 MDa / Experimental value: NO
Source (natural)Organism: Aeromonas salmonicida (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2150 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
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 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1Warpparticle selection
2Topazparticle selection
3SerialEMimage acquisition
5WarpCTF correction
8UCSF Chimeramodel fitting
10cryoSPARCinitial Euler assignment
11RELIONfinal Euler assignment
14Cootmodel refinement
15RosettaEMmodel refinement
16PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53353 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingPDB-ID: 6PIF

6pif

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