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- EMDB-19284: Conformational Landscape of the Type V-K CRISPR-associated Transp... -
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Open data
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Basic information
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Title | Conformational Landscape of the Type V-K CRISPR-associated TransposonIntegration Assembly CAST V-K TnsB domain local-refinement map | |||||||||
![]() | CAST V-K TnsB domain local-refinement map | |||||||||
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![]() | CRISPR-associated Transposon genome editing transposition / DNA BINDING PROTEIN | |||||||||
Function / homology | ![]() | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.11 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 | ![]() | 98.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 22.9 KB 22.9 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 28.7 KB | Display | ![]() |
Images | ![]() | 152.3 KB | ||
Masks | ![]() | 106.1 MB | ![]() | |
Filedesc metadata | ![]() | 6.8 KB | ||
Others | ![]() ![]() | 98.6 MB 98.6 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8rkvMC ![]() 8axaC ![]() 8rduC ![]() 8rktC ![]() 8rkuC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||
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Annotation | CAST V-K TnsB domain local-refinement 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 TnsB domain local refinement, Half-B map
File | emd_19284_half_map_1.map | ||||||||||||
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Annotation | CAST V-K TnsB domain local refinement, Half-B map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: CAST V-K TnsB domain local refinement, Half-A map
File | emd_19284_half_map_2.map | ||||||||||||
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Annotation | CAST V-K TnsB domain local refinement, Half-A 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...
Entire | Name: Type V-K CRISPR-associated transposon post-transposition state after transesterification (TnsB domain) |
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Components |
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-Supramolecule #1: Type V-K CRISPR-associated transposon post-transposition state af...
Supramolecule | Name: Type V-K CRISPR-associated transposon post-transposition state after transesterification (TnsB domain) type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 1 MDa |
-Macromolecule #1: Non-target strand - LE
Macromolecule | Name: Non-target strand - LE / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 20.961504 KDa |
Sequence | String: (DG)(DT)(DG)(DA)(DA)(DG)(DG)(DT)(DT)(DC) (DT)(DC)(DT)(DT)(DC)(DA)(DG)(DT)(DA)(DT) (DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG)(DC) (DC)(DA)(DC)(DT)(DG)(DT)(DT)(DA)(DA)(DA) (DA) (DC)(DG)(DT)(DA)(DC)(DT) ...String: (DG)(DT)(DG)(DA)(DA)(DG)(DG)(DT)(DT)(DC) (DT)(DC)(DT)(DT)(DC)(DA)(DG)(DT)(DA)(DT) (DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG)(DC) (DC)(DA)(DC)(DT)(DG)(DT)(DT)(DA)(DA)(DA) (DA) (DC)(DG)(DT)(DA)(DC)(DT)(DA)(DT) (DA)(DT)(DA)(DG)(DA)(DC)(DA)(DT)(DC)(DT) (DC)(DC) (DA)(DC)(DA)(DA)(DA)(DA)(DG) (DG) |
-Macromolecule #2: Target strand - LE
Macromolecule | Name: Target strand - LE / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 40.82216 KDa |
Sequence | String: (DA)(DA)(DT)(DT)(DA)(DA)(DA)(DT)(DA)(DG) (DT)(DC)(DA)(DC)(DA)(DA)(DT)(DG)(DA)(DC) (DA)(DT)(DT)(DA)(DA)(DT)(DC)(DT)(DG) (DT)(DC)(DA)(DC)(DC)(DG)(DA)(DC)(DG)(DA) (DC) (DA)(DG)(DA)(DT)(DA)(DA) ...String: (DA)(DA)(DT)(DT)(DA)(DA)(DA)(DT)(DA)(DG) (DT)(DC)(DA)(DC)(DA)(DA)(DT)(DG)(DA)(DC) (DA)(DT)(DT)(DA)(DA)(DT)(DC)(DT)(DG) (DT)(DC)(DA)(DC)(DC)(DG)(DA)(DC)(DG)(DA) (DC) (DA)(DG)(DA)(DT)(DA)(DA)(DT)(DT) (DT)(DG)(DT)(DC)(DA)(DC)(DT)(DG)(DT)(DA) (DC)(DA) (DC)(DT)(DA)(DC)(DG)(DC)(DC) (DT)(DT)(DT)(DT)(DG)(DT)(DG)(DG)(DA)(DG) (DA)(DT)(DG) (DT)(DC)(DT)(DA)(DA)(DT) (DA)(DT)(DC)(DT)(DA)(DC)(DG)(DT)(DT)(DT) (DT)(DA)(DA)(DC) (DA)(DG)(DT)(DG)(DG) (DC)(DC)(DT)(DT)(DA)(DT)(DT)(DA)(DA)(DA) (DT)(DG)(DA)(DC)(DT) (DT)(DC)(DT)(DC) (DA)(DA)(DC)(DC)(DT)(DT)(DC)(DA)(DC) |
-Macromolecule #3: LE
Macromolecule | Name: LE / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 23.238008 KDa |
Sequence | String: (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DA)(DC) (DA)(DG)(DT)(DG)(DA)(DC)(DA)(DA)(DA) (DT)(DT)(DA)(DT)(DC)(DT)(DG)(DT)(DC)(DG) (DT) (DC)(DG)(DG)(DT)(DG)(DA) ...String: (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DA)(DC) (DA)(DG)(DT)(DG)(DA)(DC)(DA)(DA)(DA) (DT)(DT)(DA)(DT)(DC)(DT)(DG)(DT)(DC)(DG) (DT) (DC)(DG)(DG)(DT)(DG)(DA)(DC)(DA) (DG)(DA)(DT)(DT)(DA)(DA)(DT)(DG)(DT)(DC) (DA)(DT) (DT)(DG)(DT)(DG)(DA)(DC)(DT) (DA)(DT)(DT)(DT)(DA)(DA)(DT)(DT) |
-Macromolecule #4: RE
Macromolecule | Name: RE / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 22.876809 KDa |
Sequence | String: (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DA)(DC) (DA)(DG)(DT)(DG)(DA)(DC)(DT)(DA)(DA) (DT)(DT)(DA)(DT)(DA)(DT)(DG)(DT)(DC)(DG) (DT) (DT)(DG)(DT)(DG)(DA)(DC) ...String: (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DA)(DC) (DA)(DG)(DT)(DG)(DA)(DC)(DT)(DA)(DA) (DT)(DT)(DA)(DT)(DA)(DT)(DG)(DT)(DC)(DG) (DT) (DT)(DG)(DT)(DG)(DA)(DC)(DA)(DA) (DA)(DT)(DT)(DA)(DT)(DT)(DG)(DT)(DC)(DA) (DT)(DC) (DA)(DG)(DT)(DA)(DA)(DA)(DA) (DT)(DC)(DC)(DT)(DT)(DA)(DT) |
-Macromolecule #5: Non-target strand - RE
Macromolecule | Name: Non-target strand - RE / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 24.402717 KDa |
Sequence | String: (DA)(DT)(DA)(DA)(DG)(DG)(DA)(DT)(DT)(DT) (DT)(DA)(DC)(DT)(DG)(DA)(DT)(DG)(DA)(DC) (DA)(DA)(DT)(DA)(DA)(DT)(DT)(DT)(DG) (DT)(DC)(DA)(DC)(DA)(DA)(DC)(DG)(DA)(DC) (DA) (DT)(DA)(DT)(DA)(DA)(DT) ...String: (DA)(DT)(DA)(DA)(DG)(DG)(DA)(DT)(DT)(DT) (DT)(DA)(DC)(DT)(DG)(DA)(DT)(DG)(DA)(DC) (DA)(DA)(DT)(DA)(DA)(DT)(DT)(DT)(DG) (DT)(DC)(DA)(DC)(DA)(DA)(DC)(DG)(DA)(DC) (DA) (DT)(DA)(DT)(DA)(DA)(DT)(DT)(DA) (DG)(DT)(DC)(DA)(DC)(DT)(DG)(DT)(DA)(DC) (DA)(DC) (DG)(DT)(DA)(DG)(DA)(DG)(DA) (DC)(DG)(DT)(DA)(DG)(DC)(DA)(DA)(DT)(DG) (DC)(DT) |
-Macromolecule #6: Target strand
Macromolecule | Name: Target strand / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 4.559971 KDa |
Sequence | String: (DA)(DG)(DC)(DA)(DT)(DT)(DG)(DC)(DT)(DA) (DC)(DG)(DT)(DC)(DT) |
-Macromolecule #7: TnsB
Macromolecule | Name: TnsB / type: protein_or_peptide / ID: 7 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 66.592945 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: SNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQD GLVGLTQTSR ADKGKHRIGE FWENFITKTY KEGNKGSKRM TPKQVALRVE AKARELKDSK PPNYKTVLRV L APILEKQQ ...String: SNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQD GLVGLTQTSR ADKGKHRIGE FWENFITKTY KEGNKGSKRM TPKQVALRVE AKARELKDSK PPNYKTVLRV L APILEKQQ KAKSIRSPGW RGTTLSVKTR EGKDLSVDYS NHVWQCDHTR VDVLLVDQHG EILSRPWLTT VIDTYSRCIM GI NLGFDAP SSGVVALALR HAILPKRYGS EYKLHCEWGT YGKPEHFYTD GGKDFRSNHL SQIGAQLGFV CHLRDRPSEG GVV ERPFKT LNDQLFSTLP GYTGSNVQER PEDAEKDARL TLRELEQLLV RYIVDRYNQS IDARMGDQTR FERWEAGLPT VPVP IPERD LDICLMKQSR RTVQRGGCLQ FQNLMYRGEY LAGYAGETVN LRFDPRDITT ILVYRQENNQ EVFLTRAHAQ GLETE QLAL DEAEAASRRL RTAGKTISNQ SLLQEVVDRD ALVATKKSRK ERQKLEQTVL RSAAVDESNR ESLPSQIVEP DEVEST ETV HSQYEDIEVW DYEQLREEYG F UniProtKB: TnsB |
-Macromolecule #8: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-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) / Number real images: 19936 / 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
Refinement | Protocol: AB INITIO MODEL |
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Output model | ![]() PDB-8rkv: |