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- EMDB-52109: SV40 LTAg complex with ADP and DNA (3D variability component_000,... -

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Basic information

Entry
Database: EMDB / ID: EMD-52109
TitleSV40 LTAg complex with ADP and DNA (3D variability component_000, frame_019).
Map dataIntermediate map from continuous heterogeneity analysis, postprocessed by EMReady2. Frame_019 (of frames _000 thru _019) along principal component_000 (of components _000, 001, & _002).
Sample
  • Complex: Large T antigen with DNA in presence of ADP.
    • Complex: Large T antigen
    • Complex: DNA
KeywordsAAA+ superfamily Helicase Substrate translocation DNA unwinding / DNA BINDING PROTEIN
Biological speciesBetapolyomavirus macacae / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsShahid T
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_PC_17136 United Kingdom
Other private
CitationJournal: Nature / Year: 2025
Title: Structural dynamics of DNA unwinding by a replicative helicase.
Authors: Taha Shahid / Ammar U Danazumi / Muhammad Tehseen / Lubna Alhudhali / Alice R Clark / Christos G Savva / Samir M Hamdan / Alfredo De Biasio /
Abstract: Hexameric helicases are nucleotide-driven molecular machines that unwind DNA to initiate replication across all domains of life. Despite decades of intensive study, several critical aspects of their ...Hexameric helicases are nucleotide-driven molecular machines that unwind DNA to initiate replication across all domains of life. Despite decades of intensive study, several critical aspects of their function remain unresolved: the site and mechanism of DNA strand separation, the mechanics of unwinding propagation, and the dynamic relationship between nucleotide hydrolysis and DNA movement. Here, using cryo-electron microscopy (cryo-EM), we show that the simian virus 40 large tumour antigen (LTag) helicase assembles in the form of head-to-head hexamers at replication origins, melting DNA at two symmetrically positioned sites to establish bidirectional replication forks. Through continuous heterogeneity analysis, we characterize the conformational landscape of LTag on forked DNA under catalytic conditions, demonstrating coordinated motions that drive DNA translocation and unwinding. We show that the helicase pulls the tracking strand through DNA-binding loops lining the central channel, while directing the non-tracking strand out of the rear, in a cyclic process. ATP hydrolysis functions as an 'entropy switch', removing blocks to translocation rather than directly powering DNA movement. Our structures show the allosteric couplings between nucleotide turnover and subunit motions that enable DNA unwinding while maintaining dedicated exit paths for the separated strands. These findings provide a comprehensive model for replication fork establishment and progression that extends from viral to eukaryotic systems. More broadly, they introduce fundamental principles of the mechanism by which ATP-dependent enzymes achieve efficient mechanical work through entropy-driven allostery.
History
DepositionNov 17, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52109.png

Downloads & links

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Map

FileDownload / File: emd_52109.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIntermediate map from continuous heterogeneity analysis, postprocessed by EMReady2. Frame_019 (of frames _000 thru _019) along principal component_000 (of components _000, 001, & _002).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.67 Å/pix.
x 192 pix.
= 320.64 Å		1.67 Å/pix.
x 192 pix.
= 320.64 Å		1.67 Å/pix.
x 192 pix.
= 320.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.67 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.3
Minimum - Maximum-0.08143592 - 12.462949
Average (Standard dev.)0.03550121 (±0.45282042)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 320.63998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Unfiltered and unmasked gold-standard half-map (consensus refinement)....

Fileemd_52109_half_map_1.map
AnnotationUnfiltered and unmasked gold-standard half-map (consensus refinement).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered and unmasked gold-standard half-map (consensus refinement)....

Fileemd_52109_half_map_2.map
AnnotationUnfiltered and unmasked gold-standard half-map (consensus refinement).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Large T antigen with DNA in presence of ADP.

EntireName: Large T antigen with DNA in presence of ADP.
Components
  • Complex: Large T antigen with DNA in presence of ADP.
    • Complex: Large T antigen
    • Complex: DNA

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Supramolecule #1: Large T antigen with DNA in presence of ADP.

SupramoleculeName: Large T antigen with DNA in presence of ADP. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Large T antigen

SupramoleculeName: Large T antigen / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Betapolyomavirus macacae

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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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

Concentration0.1 mg/mL
BufferpH: 8 / Details: 200 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1 mM ADP.
GridModel: UltrAuFoil / Material: GOLD / Support film - Material: GRAPHENE OXIDE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 9737 / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 97587
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC

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