+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1685 | |||||||||
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Title | Double hexamer of LTag108-627 mutant | |||||||||
Map data | Double hexamer of LTag108-627 mutant | |||||||||
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: 25.0 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_1685.map.gz | 1.6 MB | EMDB map data format | |
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Header (meta data) | emd-1685-v30.xml emd-1685.xml | 10.3 KB 10.3 KB | Display Display | EMDB header |
Images | emd_1685.jpg | 26.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1685 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1685 | HTTPS FTP |
-Validation report
Summary document | emd_1685_validation.pdf.gz | 213.2 KB | Display | EMDB validaton report |
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Full document | emd_1685_full_validation.pdf.gz | 212.3 KB | Display | |
Data in XML | emd_1685_validation.xml.gz | 5.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1685 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1685 | HTTPS FTP |
-Related structure data
Related structure data | 1681C 1682C 1683C 1684C 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_1685.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Double hexamer of LTag108-627 mutant | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.2 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 LTag108-627 mutant
Entire | Name: Double hexamer of LTag108-627 mutant |
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Components |
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-Supramolecule #1000: Double hexamer of LTag108-627 mutant
Supramolecule | Name: Double hexamer of LTag108-627 mutant / type: sample / ID: 1000 / Oligomeric state: Dodecameric / Number unique components: 1 |
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Molecular weight | Experimental: 800 KDa / Theoretical: 800 KDa |
-Macromolecule #1: LTag108-627
Macromolecule | Name: LTag108-627 / type: protein_or_peptide / ID: 1 / Name.synonym: LTag108-627 / Number of copies: 12 / Oligomeric state: Dodecameric / Recombinant expression: Yes |
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Source (natural) | Organism: Simian virus 40 / synonym: SV49 |
Molecular weight | Experimental: 800 KDa / Theoretical: 800 KDa |
-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 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.1 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 seconds 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: 60 / 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: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Xmipp / Number images used: 9000 |
Final two d classification | Number classes: 211 |