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- PDB-9kae: CryoEM structure of LTag bound to SV40 EP half origin DNA -

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Basic information

Entry
Database: PDB / ID: 9kae
TitleCryoEM structure of LTag bound to SV40 EP half origin DNA
Components
  • (DNA) x 2
  • Large T antigen
KeywordsTRANSLOCASE / Helicase / ATPase / EP half origin / AMP-PNP / DNA replication
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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Large T antigen
Similarity search - Component
Biological speciesBetapolyomavirus macacae
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsDanazumi, A.U. / Shahid, T. / Tehseen, M. / Alhudhali, L. / Clark, A. / Savva, C.G. / Hamdan, S.M. / De Biasio, A.
Funding support Saudi Arabia, 1items
OrganizationGrant numberCountry
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
DepositionOct 28, 2024Deposition site: PDBJ / Processing site: PDBJ
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.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Apr 2, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _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
P: DNA
T: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,66420
Polymers258,4818
Non-polymers3,18312
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Large T antigen / LT / LT-AG / DNA 3'-5' helicase large T antigen


Mass: 41812.664 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Betapolyomavirus macacae / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03070, DNA 3'-5' helicase
#2: DNA chain DNA


Mass: 3087.110 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA


Mass: 4517.935 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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
1Complex of SV40 LTag with SV40 EP half originCOMPLEX#1-#30MULTIPLE SOURCES
2SV40 LTagCOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Betapolyomavirus macacae1891767
23synthetic construct (others)32630
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1100 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: 5 sec. / Electron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 148881 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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