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- PDB-8bd4: TniQ-capped Tns-ATP-dsDNA complex -

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Basic information

Entry
Database: PDB / ID: 8bd4
TitleTniQ-capped Tns-ATP-dsDNA complex
Components
  • DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')
  • TniQ (Homology model)
  • TnsC
KeywordsDNA BINDING PROTEIN / Transposition / TniQ / TnsC / CRISPR-Cas / Tn7-like transposon
Function / homology
Function and homology information


Bacterial TniB / Bacterial TniB protein / TniQ / TniQ / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / TnsC / TniQ (Homology model)
Similarity search - Component
Biological speciesScytonema hofmannii (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsQuerques, I. / Schmitz, M. / Oberli, S. / Chanez, C. / Jinek, M.
Funding supportEuropean Union, Switzerland, United States, 4items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 4, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TnsC
B: TnsC
C: TnsC
D: TnsC
E: TnsC
F: TnsC
G: TnsC
R: TniQ (Homology model)
S: TniQ (Homology model)
T: TniQ (Homology model)
U: DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')
V: DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)291,05532
Polymers286,94212
Non-polymers4,11320
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 10 molecules ABCDEFGRST

#1: Protein
TnsC


Mass: 31444.617 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8J0PCL3
#2: Protein TniQ (Homology model)


Mass: 19011.240 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8J0PCL5

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DNA chain , 1 types, 2 molecules UV

#3: DNA chain DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')


Mass: 4898.191 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 20 molecules

#4: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of TnsC and TniQ transposon proteins bound to ATP and dsDNA
Type: COMPLEX / Details: TniQ, TnsC, dsDNA / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 66.036 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35964 / Symmetry type: POINT

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