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- EMDB-19283: Conformational Landscape of the Type V-K CRISPR-associated Transp... -

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Basic information

Entry
Database: EMDB / ID: EMD-19283
TitleConformational Landscape of the Type V-K CRISPR-associated TransposonIntegration Assembly CAST V-K TnsC domain local-refinement map
Map dataCAST V-K TnsC domain local-refinement map
Sample
  • Complex: Type V-K CRISPR-associated transposon post-transposition state after transesterification
    • DNA: Non-target strand - LE
    • DNA: Target strand - LE
    • Protein or peptide: ShTnsC
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: water
KeywordsCRISPR-associated Transposon genome editing transposition / DNA BINDING PROTEIN
Function / homologyBacterial TniB / Bacterial TniB protein / P-loop containing nucleoside triphosphate hydrolase / TnsC
Function and homology information
Biological speciesScytonema hofmannii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsTenjo-Castano F / Mesa P / Montoya G
Funding support Denmark, European Union, 2 items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
European Research Council (ERC)101096548European Union
CitationJournal: 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
DepositionDec 30, 2023-
Header (metadata) releaseJun 19, 2024-
Map releaseJun 19, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19283.png

Downloads & links

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Map

FileDownload / File: emd_19283.map.gz / Format: CCP4 / Size: 106.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCAST V-K TnsC domain local-refinement map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 483 pix.
= 351.624 Å		0.73 Å/pix.
x 241 pix.
= 175.448 Å		0.73 Å/pix.
x 239 pix.
= 173.992 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.728 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.26
Minimum - Maximum-0.9028086 - 2.206369
Average (Standard dev.)0.0035330215 (±0.080069825)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin314315152
Dimensions241239483
Spacing239241483
CellA: 173.99199 Å / B: 175.448 Å / C: 351.624 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19283_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CAST V-K TnsC domain local refinement, half-A map

Fileemd_19283_half_map_1.map
AnnotationCAST V-K TnsC domain local refinement, half-A map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CAST V-K TnsC domain local refinement, half-B map

Fileemd_19283_half_map_2.map
AnnotationCAST V-K TnsC domain local refinement, half-B map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Type V-K CRISPR-associated transposon post-transposition state af...

EntireName: Type V-K CRISPR-associated transposon post-transposition state after transesterification
Components
  • Complex: Type V-K CRISPR-associated transposon post-transposition state after transesterification
    • DNA: Non-target strand - LE
    • DNA: Target strand - LE
    • Protein or peptide: ShTnsC
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: water

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Supramolecule #1: Type V-K CRISPR-associated transposon post-transposition state af...

SupramoleculeName: Type V-K CRISPR-associated transposon post-transposition state after transesterification
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 1 MDa

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Macromolecule #1: Non-target strand - LE

MacromoleculeName: Non-target strand - LE / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 20.961504 KDa
SequenceString: (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)

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Macromolecule #2: Target strand - LE

MacromoleculeName: Target strand - LE / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 40.82216 KDa
SequenceString: (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)

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Macromolecule #3: ShTnsC

MacromoleculeName: ShTnsC / type: protein_or_peptide / ID: 3 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 31.400496 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: STEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV ...String:
STEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV VLVGTDRLDA VIKRDEQVLE RFRAHLRFGK LSGEDFKNTV EMWEQMVLKL PVSSNLKSKE MLRILTSATE GY IGRLDEI LREAAIRSLS RGLKKIDKAV LQEVAKEYK

UniProtKB: TnsC

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 14 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 14 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 538 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridModel: UltrAuFoil R0./1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Specialist opticsEnergy filter - Name: TFS Selectris X
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 19936 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 258000
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementProtocol: AB INITIO MODEL
Output model

PDB-8rku:
Conformational Landscape of the Type V-K CRISPR-associated TransposonIntegration Assembly CAST V-K TnsC domain local-refinement map

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