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- PDB-9f75: Active SV40 LTAg complex with DNA (3D variability component_000, ... -

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Basic information

Entry
Database: PDB / ID: 9f75
TitleActive SV40 LTAg complex with DNA (3D variability component_000, frame_019).
Components
  • Chains: S
  • Large T antigen
KeywordsDNA BINDING PROTEIN / AAA+ superfamily Helicase Substrate translocation DNA unwinding
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 / helicase activity / isomerase activity / single-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 / helicase activity / isomerase activity / single-stranded DNA binding / double-stranded DNA binding / symbiont-mediated perturbation of host ubiquitin-like protein modification / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / DNA clamp loader activity / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated suppression of host innate immune response / : / host cell nucleus / ATP hydrolysis activity / ATP binding / identical protein binding / metal ion 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
ADENOSINE-5'-TRIPHOSPHATE / DNA / Large T antigen
Similarity search - Component
Biological speciesBetapolyomavirus macacae
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsShahid, T.
Funding support United Kingdom, 2items
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
DepositionMay 3, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release
Revision 1.1Mar 26, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Apr 2, 2025Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Large T antigen
B: Large T antigen
C: Large T antigen
D: Large T antigen
E: Large T antigen
F: Large T antigen
S: Chains: S
hetero molecules


Theoretical massNumber of molelcules
Total (without water)256,30813
Polymers253,2657
Non-polymers3,0436
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Large T antigen / LT / LT-AG


Mass: 41812.664 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Betapolyomavirus macacae / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P03070, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: DNA chain Chains: S


Mass: 2388.585 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Forked DNA. Annealed product of oligonucleotides TTCTGTGACTACCTGGACGACCGGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT & TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCCGGTCGTCCAGGTAGTCACAGA.
Source: (synth.) synthetic construct (others)
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1SV40 large T antigen complex with DNA in presence of ATP and Mg2+.COMPLEX#1-#20MULTIPLE SOURCES
2Large T antigenCOMPLEX#11RECOMBINANT
3DNACOMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Betapolyomavirus macacae1891767
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
22Spodoptera frugiperda (fall armyworm)307454Sf9
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Details: 50 mM HEPES (pH 7.5), 1 mM ATP, 3 mM MgCl2, 1 mM DTT and 50 mM NaCl.
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 40 mA / Grid material: GOLD / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 8460
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1Topazparticle selection
2EPUimage acquisition
4CTFFIND4CTF correction
5RELION4CTF correction
6cryoSPARCCTF correction
9iMODFITmodel fitting
10ISOLDEmodel fitting
12ISOLDEmodel refinement
13PHENIXmodel refinement
14cryoSPARCinitial Euler assignment
15cryoSPARCfinal Euler assignmentNon-uniform refinement
16cryoSPARCclassification
17cryoSPARC3D reconstructionNon-uniform refinement
18cryoSPARC3D reconstruction3DVA
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 201416 / Details: Consensus refinement. / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building
ID 3D fitting-IDDetailsSource nameType
11"Static" LTAg-ATP-DNA model (to be separately deposited)Otherexperimental model
21AlphaFoldin silico model

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