+Open data
-Basic information
Entry | Database: PDB / ID: 7ufm | ||||||
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Title | VchTnsC AAA+ with DNA (double heptamer) | ||||||
Components |
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Keywords | DNA BINDING PROTEIN/DNA / transposon / AAA+ / DNA BINDING PROTEIN-DNA complex | ||||||
Function / homology | ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) Function and homology information | ||||||
Biological species | Vibrio cholerae (bacteria) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||
Authors | Fernandez, I.S. / Sternberg, S.H. | ||||||
Funding support | United States, 1items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7ufm.cif.gz | 768.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ufm.ent.gz | 648.5 KB | Display | PDB format |
PDBx/mmJSON format | 7ufm.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7ufm_validation.pdf.gz | 2.1 MB | Display | wwPDB validaton report |
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Full document | 7ufm_full_validation.pdf.gz | 2.2 MB | Display | |
Data in XML | 7ufm_validation.xml.gz | 129.4 KB | Display | |
Data in CIF | 7ufm_validation.cif.gz | 168.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/uf/7ufm ftp://data.pdbj.org/pub/pdb/validation_reports/uf/7ufm | HTTPS FTP |
-Related structure data
Related structure data | 26477MC 7rzyC 7ufiC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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 | | Mass: 11055.130 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #3: DNA chain | | Mass: 11099.121 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #4: Chemical | ChemComp-ATP / Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: VchTnsC AAA+ with DNA (double heptamer) / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES |
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Source (natural) | Organism: Vibrio cholerae (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.2 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Homemade |
Vitrification | Cryogen name: NITROGEN |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: NONE |
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3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 125000 / Algorithm: FOURIER SPACE / Symmetry type: POINT |
Atomic model building | Protocol: OTHER / Space: REAL |