+Open data
-Basic information
Entry | Database: PDB / ID: 7u5e | |||||||||
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Title | I-F3b Cascade-TniQ partial R-loop complex | |||||||||
Components |
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Keywords | DNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas / Transposon / CAST / Cascade / I-F / I-F3 / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex | |||||||||
Function / homology | DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) Function and homology information | |||||||||
Biological species | Aeromonas salmonicida (bacteria) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.03 Å | |||||||||
Authors | Park, J.U. / Mehrotra, E. / Kellogg, E.H. | |||||||||
Funding support | United States, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7u5e.cif.gz | 645.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7u5e.ent.gz | 505.6 KB | Display | PDB format |
PDBx/mmJSON format | 7u5e.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7u5e_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 7u5e_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 7u5e_validation.xml.gz | 98 KB | Display | |
Data in CIF | 7u5e_validation.cif.gz | 148.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/u5/7u5e ftp://data.pdbj.org/pub/pdb/validation_reports/u5/7u5e | HTTPS FTP |
-Related structure data
Related structure data | 26349MC 7u5dC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-RNA chain , 1 types, 1 molecules 1
#1: RNA chain | 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 | Mass: 35613.680 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Aeromonas salmonicida (bacteria) |
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#3: DNA chain | Mass: 35737.723 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Aeromonas salmonicida (bacteria) |
-Protein , 4 types, 10 molecules ABCDEFGHIJ
#4: Protein | 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) | ||||
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#5: Protein | 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 | | 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 | 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) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: I-F3b Cascade-TniQ Full R-loop complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 0.45 MDa / Experimental value: NO | |||||||||||||||
Source (natural) | Organism: Aeromonas salmonicida (bacteria) | |||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | |||||||||||||||
Buffer solution | pH: 7.5 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34800 / Symmetry type: POINT | ||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL | ||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6PIF Accession code: 6PIF / Source name: PDB / Type: experimental model |