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- EMDB-25453: TnsBctd-TnsC-TniQ complex -

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Basic information

Entry
Database: EMDB / ID: EMD-25453
TitleTnsBctd-TnsC-TniQ complex
Map dataLocSpiral processed map
Sample
  • Complex: ATP-bound TnsBctd-TnsC-TniQ complex from ShCAST element
    • DNA: DNA (28-MER)
    • DNA: DNA (29-MER)
    • Protein or peptide: TnsC
    • Protein or peptide: TnsB-CTD
    • Protein or peptide: TniQ
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
Biological species[Scytonema hofmanni] UTEX 2349 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsPark J / Tsai AWT / Kellogg EH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00-GM124463 United States
CitationJournal: Nature / Year: 2023
Title: Structures of the holo CRISPR RNA-guided transposon integration complex.
Authors: Jung-Un Park / Amy Wei-Lun Tsai / Alexandrea N Rizo / Vinh H Truong / Tristan X Wellner / Richard D Schargel / Elizabeth H Kellogg /
Abstract: CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR ...CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
History
DepositionNov 19, 2021-
Header (metadata) releaseNov 23, 2022-
Map releaseNov 23, 2022-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25453.png

Downloads & links

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Map

FileDownload / File: emd_25453.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocSpiral processed map
Voxel sizeX=Y=Z: 1.33 Å
Density
Contour LevelBy AUTHOR: 3.5
Minimum - Maximum0.0 - 21.737415
Average (Standard dev.)0.1757795 (±0.6893291)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 340.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25453_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full map

Fileemd_25453_additional_1.map
AnnotationFull map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: uniform B-factor sharpened map

Fileemd_25453_additional_2.map
Annotationuniform B-factor sharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap1

Fileemd_25453_half_map_1.map
Annotationhalfmap1
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: halfmap 2

Fileemd_25453_half_map_2.map
Annotationhalfmap 2
Projections & Slices
AxesZYX

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Sample components

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Entire : ATP-bound TnsBctd-TnsC-TniQ complex from ShCAST element

EntireName: ATP-bound TnsBctd-TnsC-TniQ complex from ShCAST element
Components
  • Complex: ATP-bound TnsBctd-TnsC-TniQ complex from ShCAST element
    • DNA: DNA (28-MER)
    • DNA: DNA (29-MER)
    • Protein or peptide: TnsC
    • Protein or peptide: TnsB-CTD
    • Protein or peptide: TniQ
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: ATP-bound TnsBctd-TnsC-TniQ complex from ShCAST element

SupramoleculeName: ATP-bound TnsBctd-TnsC-TniQ complex from ShCAST element
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: DNA (28-MER)

MacromoleculeName: DNA (28-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.472441 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #2: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.03804 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)

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Macromolecule #3: TnsC

MacromoleculeName: TnsC / type: protein_or_peptide / ID: 3 / Number of copies: 11 / Enantiomer: LEVO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Molecular weightTheoretical: 31.444617 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV ...String:
MTEAQAIAKQ 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

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Macromolecule #4: TnsB-CTD

MacromoleculeName: TnsB-CTD / type: protein_or_peptide / ID: 4 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Molecular weightTheoretical: 1.97711 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
IEVWDYEQLR EEYGF

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Macromolecule #5: TniQ

MacromoleculeName: TniQ / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Molecular weightTheoretical: 19.01124 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MIEAPDVKPW LFLIKPYEGE SLSHFLGRFR RANHLSASGL GTLAGIGAIV ARWERFHFNP RPSQQELEAI ASVVEVDAQR LAQMLPPAG VGMQHEPIRL CGACYAESPC HRIEWQYKSV WKCDRHQLKI LAKCPNCQAP FKMPALWEDG CCHRCRMPFA E MAKLQKV

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 11 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 11 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
2.0 mMC10H16N5O13P3ATPAdenosine triphosphate
200.0 mMNaClSodium chlorideSodium Chloride
2.0 mMMgCl2Magnesium Chloride
2.0 %C3H8O3Glycerol
1.0 mMC4H10O2S2DTT
GridModel: UltrAuFoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

23726

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 61515
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7svu:
TnsBctd-TnsC-TniQ complex

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