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- EMDB-1687: EP helicase hexamer from Double hexamer of LTag108-627 mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-1687
TitleEP helicase hexamer from Double hexamer of LTag108-627 mutant
Map dataEP Helicase hexamer from double hexamer LTag108-627 mutant
Sample
  • Sample: EP helicase hexamer from double hexamer of LTag108-627 mutant
  • Protein or peptide: LTag108-627
  • DNA: SV40 ori
KeywordsHelicase / Large T antigen / DNA Replication / SV40
Biological speciesSimian virus 40
Methodsingle particle reconstruction / cryo EM / Resolution: 19.0 Å
AuthorsCuesta I / Nunez-Ramirez R / Scheres SHW / Gai D / Chen XS / Fanning E / Carazo JM
CitationJournal: 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
DepositionJan 22, 2010-
Header (metadata) releaseMar 3, 2010-
Map releaseJan 28, 2011-
UpdateJan 28, 2011-
Current statusJan 28, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.35
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.35
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_1687.jpg

Downloads & links

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Map

FileDownload / File: emd_1687.map.gz / Format: CCP4 / Size: 245.1 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEP Helicase hexamer from double hexamer LTag108-627 mutant
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.2 Å/pix.
x 40 pix.
= 42. Å		4.2 Å/pix.
x 40 pix.
= 42. Å		4.2 Å/pix.
x 40 pix.
= 42. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.35 / Movie #1: 1.35
Minimum - Maximum-1.77208 - 4.14357
Average (Standard dev.)0.000000000174215 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions404040
Spacing404040
CellA=B=C: 42 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.24.24.2
M x/y/z101010
origin x/y/z0.0000.0000.000
length x/y/z42.00042.00042.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ121121121
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS404040
D min/max/mean-1.7724.1440.000

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Supplemental data

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Sample components

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Entire : EP helicase hexamer from double hexamer of LTag108-627 mutant

EntireName: EP helicase hexamer from double hexamer of LTag108-627 mutant
Components
  • Sample: EP helicase hexamer from double hexamer of LTag108-627 mutant
  • Protein or peptide: LTag108-627
  • DNA: SV40 ori

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Supramolecule #1000: EP helicase hexamer from double hexamer of LTag108-627 mutant

SupramoleculeName: EP helicase hexamer from double hexamer of LTag108-627 mutant
type: sample / ID: 1000 / Oligomeric state: Hexameric / Number unique components: 1
Molecular weightExperimental: 400 KDa / Theoretical: 400 KDa

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Macromolecule #1: LTag108-627

MacromoleculeName: LTag108-627 / type: protein_or_peptide / ID: 1 / Name.synonym: LTag108-627 / Number of copies: 12 / Oligomeric state: Dodecameric / Recombinant expression: Yes
Source (natural)Organism: Simian virus 40 / synonym: SV40
Molecular weightExperimental: 400 KDa / Theoretical: 400 KDa

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Macromolecule #2: SV40 ori

MacromoleculeName: SV40 ori / type: dna / ID: 2 / Name.synonym: SV40 ori / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: Simian virus 40 / synonym: SV40
Molecular weightExperimental: 70 KDa / Theoretical: 70 KDa
SequenceString:
aagctttctc actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atgggtcgac cggatccccg

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.1 mg/mL
BufferpH: 7.5
Details: 20 mM TrisHCl (pH 7.5), 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, 3 mM ADP
GridDetails: Quantifoil grids
VitrificationCryogen name: ETHANE / Chamber temperature: 93 K / Instrument: LEICA PLUNGER / Details: Vitrification instrument: Leica Plunger / Method: Blot for 2-3 second before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 93 K / Max: 93 K / Average: 93 K
Alignment procedureLegacy - Astigmatism: CTF correction
DateNov 26, 2007
Image recordingCategory: 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 min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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 stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Detailssymmetry C6 was applied in 3D reconstruction. Tilt angle restricted to 60-90 degrees
CTF correctionDetails: Plate
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Xmipp / Number images used: 9000
Final two d classificationNumber classes: 26

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