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- EMDB-50287: SV40 large T antigen assembly with DNA in presence of ATP. -

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Basic information

Entry
Database: EMDB / ID: EMD-50287
TitleSV40 large T antigen assembly with DNA in presence of ATP.
Map dataLocScale post-processed map.
Sample
  • Complex: Large T antigen with DNA in presence of ATP.
    • Complex: Large T antigen
      • Protein or peptide: Large T antigen
    • Complex: DNA
      • DNA: Chains: P
      • DNA: Chains: T
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsAAA+ superfamily Helicase Substrate translocation DNA unwinding / DNA BINDING PROTEIN
Function / homology
Function and homology information


symbiont-mediated suppression of host JAK-STAT cascade via inhibition of JAK1 activity / bidirectional double-stranded viral DNA replication / viral DNA genome replication / DNA 3'-5' helicase / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / DNA replication origin binding / isomerase activity / helicase activity / single-stranded DNA binding / double-stranded DNA binding ...symbiont-mediated suppression of host JAK-STAT cascade via inhibition of JAK1 activity / bidirectional double-stranded viral DNA replication / viral DNA genome replication / DNA 3'-5' helicase / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / DNA replication origin binding / isomerase activity / helicase activity / single-stranded DNA binding / double-stranded DNA binding / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA replication / symbiont-mediated suppression of host innate immune response / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell nucleus / ATP hydrolysis activity / zinc ion binding / ATP binding / identical protein binding
Similarity search - Function
Large T antigen, polyomaviridae / Large T antigen, polyomavirus, C-terminal / Zinc finger, large T-antigen D1-type / Origin of replication binding protein / Polyomavirus large T antigen C-terminus / Large T-antigen (T-ag) origin-binding domain (OBD) profile. / Zinc finger large T-antigen (T-ag) D1-type profile. / T antigen, Ori-binding / Zinc finger, large T-antigen D1 domain superfamily / Helicase, superfamily 3, DNA virus ...Large T antigen, polyomaviridae / Large T antigen, polyomavirus, C-terminal / Zinc finger, large T-antigen D1-type / Origin of replication binding protein / Polyomavirus large T antigen C-terminus / Large T-antigen (T-ag) origin-binding domain (OBD) profile. / Zinc finger large T-antigen (T-ag) D1-type profile. / T antigen, Ori-binding / Zinc finger, large T-antigen D1 domain superfamily / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / DnaJ molecular chaperone homology domain / dnaJ domain profile. / Chaperone J-domain superfamily / DnaJ domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesBetapolyomavirus macacae / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsShahid T / De Biasio A
Funding support United Kingdom, Saudi Arabia, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_PC_17136 United Kingdom
Other private Saudi Arabia
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
DepositionMay 11, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateMay 7, 2025-
Current statusMay 7, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50287.png

Downloads & links

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Map

FileDownload / File: emd_50287.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocScale post-processed map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 384 pix.
= 320.64 Å		0.84 Å/pix.
x 384 pix.
= 320.64 Å		0.84 Å/pix.
x 384 pix.
= 320.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.32
Minimum - Maximum-9.995685 - 36.005339999999997
Average (Standard dev.)0.09502818 (±0.9622422)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 320.63998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: EMReady post-processed map. Slight origin offset due to...

Fileemd_50287_additional_1.map
AnnotationEMReady post-processed map. Slight origin offset due to pixel size interpolation.
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_50287_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_50287_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 ATP.

EntireName: Large T antigen with DNA in presence of ATP.
Components
  • Complex: Large T antigen with DNA in presence of ATP.
    • Complex: Large T antigen
      • Protein or peptide: Large T antigen
    • Complex: DNA
      • DNA: Chains: P
      • DNA: Chains: T
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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

SupramoleculeName: Large T antigen with DNA in presence of ATP. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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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-#3
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Large T antigen

MacromoleculeName: Large T antigen / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Betapolyomavirus macacae
Molecular weightTheoretical: 41.812664 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: KQVSWKLVTE YAMETKCDDV LLLLGMYLEF QYSFEMCLKC IKKEQPSHYK YHEKHYANAA IFADSKNQKT ICQQAVDTVL AKKRVDSLQ LTREQMLTNR FNDLLDRMDI MFGSTGSADI EEWMAGVAWL HCLLPKMDSV VYDFLKCMVY NIPKKRYWLF K GPIDSGKT ...String:
KQVSWKLVTE YAMETKCDDV LLLLGMYLEF QYSFEMCLKC IKKEQPSHYK YHEKHYANAA IFADSKNQKT ICQQAVDTVL AKKRVDSLQ LTREQMLTNR FNDLLDRMDI MFGSTGSADI EEWMAGVAWL HCLLPKMDSV VYDFLKCMVY NIPKKRYWLF K GPIDSGKT TLAAALLELC GGKALNVNLP LDRLNFELGV AIDQFLVVFE DVKGTGGESR DLPSGQGINN LDNLRDYLDG SV KVNLEKK HLNKRTQIFP PGIVTMNEYS VPKTLQARFV KQIDFRPKDY LKHCLERSEF LLEKRIIQSG IALLLMLIWY RPV AEFAQS IQSRIVEWKE RLDKEFSLSV YQKMKFNVAM GIGVLD

UniProtKB: Large T antigen

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Macromolecule #2: Chains: P

MacromoleculeName: Chains: P / type: dna / ID: 2
Details: Original oligo sequence: AGACACATGGATGTAGCGAACGATCACC. Modelled as As.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.400317 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)

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Macromolecule #3: Chains: T

MacromoleculeName: Chains: T / type: dna / ID: 3
Details: Original oligo sequence: GGTGATCGTTCGC TACATGTCGTCAGGATTCCAGGCAG. Modelled as Ts.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.12632 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 6 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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 ATP.
GridModel: UltrAuFoil / Material: GOLD / Support film - Material: GRAPHENE OXIDE / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN 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

CTF correctionSoftware: (Name: CTFFIND (ver. 4), RELION (ver. 4), cryoSPARC)
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 210910
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

1svm

source_name: PDB, initial_model_type: experimental model

2gxa

chain_id: M, source_name: PDB, initial_model_type: experimental model
source_name: Other, initial_model_type: in silico modelModelAngelo
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-9fb4:
SV40 large T antigen assembly with DNA in presence of ATP.

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