+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26476 | |||||||||
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Title | VchTnsC AAA+ ATPase with DNA, single heptamer | |||||||||
Map data | post-processed, masked map | |||||||||
Sample |
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Keywords | transposon / AAA+ / DNA BINDING PROTEIN-DNA complex | |||||||||
Biological species | Vibrio cholerae (bacteria) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.4 Å | |||||||||
Authors | Fernandez IS / Sternberg SH | |||||||||
Funding support | United States, 1 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_26476.map.gz | 2 MB | EMDB map data format | |
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Header (meta data) | emd-26476-v30.xml emd-26476.xml | 21.4 KB 21.4 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_26476_fsc.xml | 7.8 KB | Display | FSC data file |
Images | emd_26476.png | 121.3 KB | ||
Masks | emd_26476_msk_1.map | 40.6 MB | Mask map | |
Filedesc metadata | emd-26476.cif.gz | 6 KB | ||
Others | emd_26476_additional_1.map.gz emd_26476_additional_2.map.gz emd_26476_half_map_1.map.gz emd_26476_half_map_2.map.gz | 3.5 MB 31.3 MB 31.4 MB 31.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26476 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26476 | HTTPS FTP |
-Validation report
Summary document | emd_26476_validation.pdf.gz | 698 KB | Display | EMDB validaton report |
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Full document | emd_26476_full_validation.pdf.gz | 697.6 KB | Display | |
Data in XML | emd_26476_validation.xml.gz | 13.5 KB | Display | |
Data in CIF | emd_26476_validation.cif.gz | 18.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26476 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26476 | HTTPS FTP |
-Related structure data
Related structure data | 7ufiMC 7rzyC 7ufmC C: citing same article (ref.) M: atomic model generated by this map |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_26476.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | post-processed, masked map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.509 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_26476_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: relion post-processed, masked map
File | emd_26476_additional_1.map | ||||||||||||
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Annotation | relion post-processed, masked map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: unsharpened map
File | emd_26476_additional_2.map | ||||||||||||
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Annotation | unsharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map 1
File | emd_26476_half_map_1.map | ||||||||||||
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Annotation | half map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map 2
File | emd_26476_half_map_2.map | ||||||||||||
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Annotation | half map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : VchTNsC AAA+ ATPase with ATP and DNA (single heptamer form)
Entire | Name: VchTNsC AAA+ ATPase with ATP and DNA (single heptamer form) |
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Components |
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-Supramolecule #1: VchTNsC AAA+ ATPase with ATP and DNA (single heptamer form)
Supramolecule | Name: VchTNsC AAA+ ATPase with ATP and DNA (single heptamer form) type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: Vibrio cholerae (bacteria) |
-Macromolecule #1: DNA (5'-D(P*CP*TP*CP*CP*AP*GP*TP*AP*CP*AP*GP*CP*GP*CP*GP*GP*CP*TP...
Macromolecule | Name: DNA (5'-D(P*CP*TP*CP*CP*AP*GP*TP*AP*CP*AP*GP*CP*GP*CP*GP*GP*CP*TP*GP*AP*A)-3') type: dna / ID: 1 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 6.433163 KDa |
Sequence | String: (DC)(DT)(DC)(DC)(DA)(DG)(DT)(DA)(DC)(DA) (DG)(DC)(DG)(DC)(DG)(DG)(DC)(DT)(DG)(DA) (DA) |
-Macromolecule #2: DNA (5'-D(P*TP*TP*CP*AP*GP*CP*CP*GP*CP*GP*CP*TP*GP*TP*AP*CP*TP*GP...
Macromolecule | Name: DNA (5'-D(P*TP*TP*CP*AP*GP*CP*CP*GP*CP*GP*CP*TP*GP*TP*AP*CP*TP*GP*GP*AP*G)-3') type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 6.455159 KDa |
Sequence | String: (DT)(DT)(DC)(DA)(DG)(DC)(DC)(DG)(DC)(DG) (DC)(DT)(DG)(DT)(DA)(DC)(DT)(DG)(DG)(DA) (DG) |
-Macromolecule #3: VchTnsC
Macromolecule | Name: VchTnsC / type: protein_or_peptide / ID: 3 / Number of copies: 7 / Enantiomer: LEVO |
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Source (natural) | Organism: Vibrio cholerae (bacteria) |
Molecular weight | Theoretical: 35.065273 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: TREARISRAK RAFVSTPSVR KILSYMDRCR DLSDLESEPT CMMVYGASGV GKTTVIKKYL NQAAAAAAAG GDIIPVLHIE LPDNAKPVD AARELLVEMG DPLALYETDL ARLTKRLTEL IPAVGVKLII IDEFQHLVEE RSNRVLTQVG NWLKMILNKT K CPIVIFGM ...String: TREARISRAK RAFVSTPSVR KILSYMDRCR DLSDLESEPT CMMVYGASGV GKTTVIKKYL NQAAAAAAAG GDIIPVLHIE LPDNAKPVD AARELLVEMG DPLALYETDL ARLTKRLTEL IPAVGVKLII IDEFQHLVEE RSNRVLTQVG NWLKMILNKT K CPIVIFGM PYSKVVLQAN SQLHGRFSIQ VELRPFSYQG GRGVFKTFLE YLDKALPFEK QAGLANESLQ KKLYAFSQGN MR SLRNLIY QASIEAIDNQ HETITEEDFV FASKLTSGDK PNSWKNPFEE GVEVTEDMLR PPPKDIGWED YLRH |
-Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 7 / Formula: ATP |
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Molecular weight | Theoretical: 507.181 Da |
Chemical component information | ChemComp-ATP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.2 |
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Grid | Model: Homemade / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING |
Vitrification | Cryogen name: NITROGEN |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 65.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |