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- PDB-7ufm: VchTnsC AAA+ with DNA (double heptamer) -

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Basic information

Entry
Database: PDB / ID: 7ufm
TitleVchTnsC AAA+ with DNA (double heptamer)
Components
  • (DNA (36-MER)) x 2
  • VchTnsC
KeywordsDNA BINDING PROTEIN/DNA / transposon / AAA+ / DNA BINDING PROTEIN-DNA complex
Function / homologyADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10)
Function and homology information
Biological speciesVibrio cholerae (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsFernandez, I.S. / Sternberg, S.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2hG011650-01 United States
CitationJournal: Nature / Year: 2022
Title: Selective TnsC recruitment enhances the fidelity of RNA-guided transposition.
Authors: Florian T Hoffmann / Minjoo Kim / Leslie Y Beh / Jing Wang / Phuc Leo H Vo / Diego R Gelsinger / Jerrin Thomas George / Christopher Acree / Jason T Mohabir / Israel S Fernández / Samuel H Sternberg /
Abstract: Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site ...Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.
History
DepositionMar 22, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 7, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Sep 21, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: VchTnsC
B: VchTnsC
C: VchTnsC
D: VchTnsC
E: VchTnsC
F: VchTnsC
G: VchTnsC
H: VchTnsC
I: VchTnsC
J: VchTnsC
K: VchTnsC
L: VchTnsC
M: VchTnsC
N: VchTnsC
O: DNA (36-MER)
P: DNA (36-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)520,16930
Polymers513,06816
Non-polymers7,10114
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
VchTnsC


Mass: 35065.273 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli)
#2: DNA chain DNA (36-MER)


Mass: 11055.130 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (36-MER)


Mass: 11099.121 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
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: VchTnsC AAA+ with DNA (double heptamer) / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Vibrio cholerae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Homemade
VitrificationCryogen name: NITROGEN

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 125000 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL

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