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- EMDB-15974: TniQ-capped Tns-ATP-dsDNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-15974
TitleTniQ-capped Tns-ATP-dsDNA complex
Map data
Sample
  • Complex: Complex of TnsC and TniQ transposon proteins bound to ATP and dsDNA
    • Protein or peptide: TnsC
    • Protein or peptide: TniQ (Homology model)
    • DNA: DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
Function / homologyBacterial TniB / Bacterial TniB protein / TniQ / TniQ / P-loop containing nucleoside triphosphate hydrolase / TnsC / TniQ (Homology model)
Function and homology information
Biological speciesScytonema hofmannii (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsQuerques I / Schmitz M / Oberli S / Chanez C / Jinek M
Funding supportEuropean Union, Switzerland, United States, 4 items
OrganizationGrant numberCountry
European Research Council (ERC)820152European Union
Swiss National Science Foundation31003A_182567 Switzerland
European Molecular Biology Organization (EMBO)ALTF 296-2020European Union
Howard Hughes Medical Institute (HHMI)55008735 United States
CitationJournal: Cell / Year: 2022
Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex.
Authors: Michael Schmitz / Irma Querques / Seraina Oberli / Christelle Chanez / Martin Jinek /
Abstract: CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the ...CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads.
History
DepositionOct 18, 2022-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateJan 4, 2023-
Current statusJan 4, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15974.png

Downloads & links

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Map

FileDownload / File: emd_15974.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.65 Å
Density
Contour LevelBy AUTHOR: 0.0548
Minimum - Maximum-0.23598246 - 0.40904927
Average (Standard dev.)0.00022723203 (±0.0093433205)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 390.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_15974_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15974_half_map_2.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 : Complex of TnsC and TniQ transposon proteins bound to ATP and dsDNA

EntireName: Complex of TnsC and TniQ transposon proteins bound to ATP and dsDNA
Components
  • Complex: Complex of TnsC and TniQ transposon proteins bound to ATP and dsDNA
    • Protein or peptide: TnsC
    • Protein or peptide: TniQ (Homology model)
    • DNA: DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Complex of TnsC and TniQ transposon proteins bound to ATP and dsDNA

SupramoleculeName: Complex of TnsC and TniQ transposon proteins bound to ATP and dsDNA
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3 / Details: TniQ, TnsC, dsDNA
Source (natural)Organism: Scytonema hofmannii (bacteria)

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Macromolecule #1: TnsC

MacromoleculeName: TnsC / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (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 #2: TniQ (Homology model)

MacromoleculeName: TniQ (Homology model) / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (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 #3: DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')

MacromoleculeName: DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')
type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.898191 KDa
SequenceString:
(DG)(DA)(DT)(DC)(DG)(DA)(DT)(DC)(DG)(DA) (DT)(DC)(DG)(DA)(DT)(DC)

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 7 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

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

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 7 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.036 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7plh

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 35964
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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