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Yorodumi- EMDB-19075: Conformational Landscape of the Type V-K CRISPR-associated Transp... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-19075 | |||||||||
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Title | Conformational Landscape of the Type V-K CRISPR-associated TransposonIntegration Assembly CAST V-K composite map | |||||||||
Map data | composite map, V-K CAST complex | |||||||||
Sample |
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Keywords | CRISPR-associated Transposon genome editing transposition / DNA BINDING PROTEIN | |||||||||
Function / homology | Function and homology information DNA integration / cytosolic small ribosomal subunit / nucleic acid binding / rRNA binding / structural constituent of ribosome / translation Similarity search - Function | |||||||||
Biological species | Scytonema hofmannii (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.3 Å | |||||||||
Authors | Tenjo-Castano F / Mesa P / Montoya G | |||||||||
Funding support | Denmark, European Union, 2 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_19075.map.gz | 97.8 MB | EMDB map data format | |
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Header (meta data) | emd-19075-v30.xml emd-19075.xml | 26.7 KB 26.7 KB | Display Display | EMDB header |
Images | emd_19075.png | 104.6 KB | ||
Filedesc metadata | emd-19075.cif.gz | 8.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-19075 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-19075 | HTTPS FTP |
-Validation report
Summary document | emd_19075_validation.pdf.gz | 502.2 KB | Display | EMDB validaton report |
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Full document | emd_19075_full_validation.pdf.gz | 501.7 KB | Display | |
Data in XML | emd_19075_validation.xml.gz | 4.3 KB | Display | |
Data in CIF | emd_19075_validation.cif.gz | 4.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-19075 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-19075 | HTTPS FTP |
-Related structure data
Related structure data | 8rduMC 8axaC 8rktC 8rkuC 8rkvC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_19075.map.gz / Format: CCP4 / Size: 106.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | composite map, V-K CAST complex | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.728 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
+Entire : Type V-K CRISPR-associated transposon post-transposition state af...
+Supramolecule #1: Type V-K CRISPR-associated transposon post-transposition state af...
+Macromolecule #1: sgRNA
+Macromolecule #2: Non-target strand - LE
+Macromolecule #3: Target strand -LE
+Macromolecule #4: LE
+Macromolecule #5: RE
+Macromolecule #6: Non-target strand - RE
+Macromolecule #7: Target strand
+Macromolecule #8: ShCas12k
+Macromolecule #9: Small ribosomal subunit protein uS15
+Macromolecule #10: TniQ
+Macromolecule #11: ShTnsC
+Macromolecule #12: TnsB
+Macromolecule #13: MAGNESIUM ION
+Macromolecule #14: ZINC ION
+Macromolecule #15: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #16: water
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7 |
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Grid | Model: UltrAuFoil R0./1 / Material: GOLD / Support film - Material: CARBON |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: TFS Selectris X |
Image recording | Film or detector model: FEI FALCON IV (4k x 4k) / Number real images: 19936 / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: DARK FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: INSILICO MODEL |
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Final reconstruction | Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 258000 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |
-Atomic model buiding 1
Initial model | Chain - Source name: AlphaFold / Chain - Initial model type: in silico model |
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Refinement | Protocol: AB INITIO MODEL |
Output model | PDB-8rdu: |