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Yorodumi- PDB-8rku: Conformational Landscape of the Type V-K CRISPR-associated Transp... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8rku | |||||||||
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Title | Conformational Landscape of the Type V-K CRISPR-associated TransposonIntegration Assembly CAST V-K TnsC domain local-refinement map | |||||||||
Components |
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Keywords | DNA BINDING PROTEIN / CRISPR-associated Transposon genome editing transposition | |||||||||
Function / homology | Bacterial TniB / Bacterial TniB protein / : / P-loop containing nucleoside triphosphate hydrolase / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA (> 100) / TnsC Function and homology information | |||||||||
Biological species | Scytonema hofmannii (bacteria) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å | |||||||||
Authors | Tenjo-Castano, F. / Mesa, P. / Montoya, G. | |||||||||
Funding support | Denmark, European Union, 2items
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Citation | Journal: Mol Cell / Year: 2024 Title: Conformational landscape of the type V-K CRISPR-associated transposon integration assembly. Authors: Francisco Tenjo-Castaño / Nicholas Sofos / Luisa S Stutzke / Piero Temperini / Anders Fuglsang / Tillmann Pape / Pablo Mesa / Guillermo Montoya / Abstract: CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site ...CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8rku.cif.gz | 741.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8rku.ent.gz | 614.1 KB | Display | PDB format |
PDBx/mmJSON format | 8rku.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8rku_validation.pdf.gz | 2.2 MB | Display | wwPDB validaton report |
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Full document | 8rku_full_validation.pdf.gz | 2.2 MB | Display | |
Data in XML | 8rku_validation.xml.gz | 105 KB | Display | |
Data in CIF | 8rku_validation.cif.gz | 164.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/rk/8rku ftp://data.pdbj.org/pub/pdb/validation_reports/rk/8rku | HTTPS FTP |
-Related structure data
Related structure data | 19283MC 8axaC 8rduC 8rktC 8rkvC C: citing same article (ref.) M: map data used to model this data |
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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
-DNA chain , 2 types, 2 molecules 23
#1: DNA chain | Mass: 20961.504 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria) |
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#2: DNA chain | Mass: 40822.160 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria) |
-Protein , 1 types, 14 molecules DEFGHIJKLMNOPQ
#3: Protein | Mass: 31400.496 Da / Num. of mol.: 14 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8J0PCL3 |
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-Non-polymers , 3 types, 566 molecules
#4: Chemical | ChemComp-MG / #5: Chemical | ChemComp-ATP / #6: Water | ChemComp-HOH / | |
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-Details
Has ligand of interest | Y |
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-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: Type V-K CRISPR-associated transposon post-transposition state after transesterification Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Molecular weight | Value: 1 MDa / Experimental value: NO |
Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R0./1 |
Vitrification | Cryogen name: ETHANE |
-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: 2000 nm / Nominal defocus min: 800 nm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 19936 |
EM imaging optics | Energyfilter name: TFS Selectris X |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 258000 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL | ||||||||||||||||||||||||||||||||
Atomic model building | Source name: AlphaFold / Type: in silico model |