+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1681 | |||||||||
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Title | Parallel conformation of wild type double hexamer LTag | |||||||||
Map data | Parallel conformation of wild type double hexamer Ltag | |||||||||
Sample |
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Keywords | Helicase / Large T antigen / DNA Replication / SV40 / Electron microscopy | |||||||||
Biological species | Simian virus 40 | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 30.0 Å | |||||||||
Authors | Cuesta I / Nunez-Ramirez R / Scheres SHW / Gai D / Chen XS / Fanning E / Carazo JM | |||||||||
Citation | Journal: J Mol Biol / Year: 2010 Title: Conformational rearrangements of SV40 large T antigen during early replication events. Authors: Isabel Cuesta / Rafael Núñez-Ramírez / Sjors H W Scheres / Dahai Gai / Xiaojiang S Chen / Ellen Fanning / Jose María Carazo / Abstract: The Simian virus 40 (SV40) large tumor antigen (LTag) functions as the replicative helicase and initiator for viral DNA replication. For SV40 replication, the first essential step is the assembly of ...The Simian virus 40 (SV40) large tumor antigen (LTag) functions as the replicative helicase and initiator for viral DNA replication. For SV40 replication, the first essential step is the assembly of an LTag double hexamer at the origin DNA that will subsequently melt the origin DNA to initiate fork unwinding. In this study, we used three-dimensional cryo-electron microscopy to visualize early events in the activation of DNA replication in the SV40 model system. We obtained structures of wild-type double-hexamer complexes of LTag bound to SV40 origin DNA, to which atomic structures have been fitted. Wild-type LTag was observed in two distinct conformations: In one conformation, the central module containing the J-domains and the origin binding domains of both hexamers is a compact closed ring. In the other conformation, the central module is an open ring with a gap formed by rearrangement of the N-terminal regions of the two hexamers, potentially allowing for the passage of single-stranded DNA generated from the melted origin DNA. Double-hexamer complexes containing mutant LTag that lacks the N-terminal J-domain show the central module predominantly in the closed-ring state. Analyses of the LTag C-terminal regions reveal that the LTag hexamers bound to the A/T-rich tract origin of replication and early palindrome origin of replication elements are structurally distinct. Lastly, visualization of DNA density protruding from the LTag C-terminal domains suggests that oligomerization of the LTag complex takes place on double-stranded DNA. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1681.map.gz | 1.8 MB | EMDB map data format | |
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Header (meta data) | emd-1681-v30.xml emd-1681.xml | 10.4 KB 10.4 KB | Display Display | EMDB header |
Images | emd_1681.jpg | 27.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1681 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1681 | HTTPS FTP |
-Validation report
Summary document | emd_1681_validation.pdf.gz | 237 KB | Display | EMDB validaton report |
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Full document | emd_1681_full_validation.pdf.gz | 236.1 KB | Display | |
Data in XML | emd_1681_validation.xml.gz | 4.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1681 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1681 | HTTPS FTP |
-Related structure data
Related structure data | 1682C 1683C 1684C 1685C 1686C 1687C C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1681.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Parallel conformation of wild type double hexamer Ltag | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 5.2 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Double hexamer of wild type LTag bound to dsDNA
Entire | Name: Double hexamer of wild type LTag bound to dsDNA |
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Components |
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-Supramolecule #1000: Double hexamer of wild type LTag bound to dsDNA
Supramolecule | Name: Double hexamer of wild type LTag bound to dsDNA / type: sample / ID: 1000 / Oligomeric state: Dodecameric / Number unique components: 2 |
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Molecular weight | Experimental: 1 MDa / Theoretical: 1 MDa / Method: Gel filtration |
-Macromolecule #1: LTag
Macromolecule | Name: LTag / type: protein_or_peptide / ID: 1 / Name.synonym: LTag / Number of copies: 12 / Oligomeric state: Dodecameric / Recombinant expression: Yes |
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Source (natural) | Organism: Simian virus 40 / synonym: SV40 / Location in cell: Nucleus |
Molecular weight | Experimental: 1 MDa / Theoretical: 1 MDa |
-Macromolecule #2: SV40 ori
Macromolecule | Name: SV40 ori / type: dna / ID: 2 / Name.synonym: SV40 ori / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes |
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Source (natural) | Organism: Simian virus 40 / synonym: SV40 |
Molecular weight | Experimental: 70 KDa / Theoretical: 70 KDa |
Sequence | String: aagctttctc actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atgggtcgac cggatccccg ggaattc |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.5 Details: 20 mM TrisHCl (pH 7.5), 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, 3 mM ADP |
Grid | Details: Quantifoil grids |
Vitrification | Cryogen name: ETHANE / Chamber temperature: 93 K / Instrument: LEICA PLUNGER / Details: Vitrification instrument: Leica Plunger / Method: Blot for 2-3 second before plunging |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Min: 93 K / Max: 93 K / Average: 93 K |
Alignment procedure | Legacy - Astigmatism: CTF correction |
Date | Nov 26, 2007 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Number real images: 93 / Average electron dose: 10 e/Å2 / Bits/pixel: 8 |
Tilt angle min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Plate |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Xmipp / Number images used: 5613 |
Final two d classification | Number classes: 211 |