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- EMDB-19286: consensus map of the V-K CRISPR-associated Transposon Integration... -
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Open data
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Basic information
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Title | consensus map of the V-K CRISPR-associated Transposon Integration Assembly | |||||||||
![]() | CAST V-K R1 global refinement, consensus map used as reference for composite map. | |||||||||
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![]() | CRISPR-associated Transposon genome editing transposition / DNA BINDING PROTEIN | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.19 Å | |||||||||
![]() | Tenjo-Castano F / Mesa P / Montoya G | |||||||||
Funding support | ![]()
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![]() | ![]() 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 | ![]() | 99.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 24.2 KB 24.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 28.6 KB | Display | ![]() |
Images | ![]() | 75.9 KB | ||
Masks | ![]() | 106.1 MB | ![]() | |
Filedesc metadata | ![]() | 7 KB | ||
Others | ![]() ![]() | 98.6 MB 98.6 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 1010.6 KB | Display | ![]() |
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Full document | ![]() | 1010.1 KB | Display | |
Data in XML | ![]() | 21.1 KB | Display | |
Data in CIF | ![]() | 31.8 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | CAST V-K R1 global refinement, consensus map used as reference for composite map. | ||||||||||||||||||||
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
-Mask #1
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Projections & Slices |
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Density Histograms |
-Half map: CAST V-K R1 global refinement, Half-A map.
File | emd_19286_half_map_1.map | ||||||||||||
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Annotation | CAST V-K R1 global refinement, Half-A map. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: CAST V-K R1 global refinement, Half-B map.
File | emd_19286_half_map_2.map | ||||||||||||
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Annotation | CAST V-K R1 global refinement, Half-B map. | ||||||||||||
Projections & Slices |
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Density Histograms |
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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: Cas12k
+Macromolecule #9: S15
+Macromolecule #10: TniQ
+Macromolecule #11: TnsC
+Macromolecule #12: TnsB
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7 |
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Grid | Model: UltrAuFoil R0./1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON |
Vitrification | Cryogen name: ETHANE |
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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) / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT 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 |
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Image processing
-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 |