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- EMDB-62502: CryoEM structure of SV40 LTag bound to SV40 origin DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-62502
TitleCryoEM structure of SV40 LTag bound to SV40 origin DNA
Map data
Sample
  • Complex: Complex of SV40 LTag with SV40 origin DNA
    • Other: LTag
KeywordsHelicase / ATPase / SV40 origin DNA / AMP-PNP / DNA replication / TRANSLOCASE
Biological speciesBetapolyomavirus macacae
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsDanazumi AU / Shahid T / Tehseen M / Alhudhali L / Clark A / Savva CG / Hamdan SM / De Biasio A
Funding support Saudi Arabia, 1 items
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
DepositionNov 25, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_62502.png

Downloads & links

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Map

FileDownload / File: emd_62502.map.gz / Format: CCP4 / Size: 600.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 540 pix.
= 502.2 Å		0.93 Å/pix.
x 540 pix.
= 502.2 Å		0.93 Å/pix.
x 540 pix.
= 502.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.47615033 - 0.7974901
Average (Standard dev.)-0.0004303128 (±0.012246381)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions540540540
Spacing540540540
CellA=B=C: 502.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_62502_msk_1.map
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Half map: #2

Fileemd_62502_half_map_1.map
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Half map: #1

Fileemd_62502_half_map_2.map
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Sample components

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Entire : Complex of SV40 LTag with SV40 origin DNA

EntireName: Complex of SV40 LTag with SV40 origin DNA
Components
  • Complex: Complex of SV40 LTag with SV40 origin DNA
    • Other: LTag

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Supramolecule #1: Complex of SV40 LTag with SV40 origin DNA

SupramoleculeName: Complex of SV40 LTag with SV40 origin DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Betapolyomavirus macacae
Molecular weightTheoretical: 1 MDa

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Macromolecule #1: LTag

MacromoleculeName: LTag / type: other / ID: 1 / Classification: other
Source (natural)Organism: Betapolyomavirus macacae
SequenceString: MDKVLNREES LQLMDLLGLE RSAWGNIPLM RKAYLKKCKE FHPDKGGDEE KMKKMNTLYK KMEDGVKYA HQPDFGGFWD ATEIPTYGTD EWEQWWNAFN EENLFCSEEM PSSDDEATAD S QHSTPPKK KRKVEDPKDF PSELLSFLSH AVFSNRTLAC FAIYTTKEKA ...String:
MDKVLNREES LQLMDLLGLE RSAWGNIPLM RKAYLKKCKE FHPDKGGDEE KMKKMNTLYK KMEDGVKYA HQPDFGGFWD ATEIPTYGTD EWEQWWNAFN EENLFCSEEM PSSDDEATAD S QHSTPPKK KRKVEDPKDF PSELLSFLSH AVFSNRTLAC FAIYTTKEKA ALLYKKIMEK YS VTFISRH NSYNHNILFF LTPHRHRVSA INNYAQKLCT FSFLICKGVN KEYLMYSALT RDP FSVIEE SLPGGLKEHD FNPEEAEETK QVSWKLVTEY AMETKCDDVL LLLGMYLEFQ YSFE MCLKC IKKEQPSHYK YHEKHYANAA IFADSKNQKT ICQQAVDTVL AKKRVDSLQL TREQM LTNR FNDLLDRMDI MFGSTGSADI EEWMAGVAWL HCLLPKMDSV VYDFLKCMVY NIPKKR YWL FKGPIDSGKT TLAAALLELC GGKALNVNLP LDRLNFELGV AIDQFLVVFE DVKGTGG ES RDLPSGQGIN NLDNLRDYLD GSVKVNLEKK HLNKRTQIFP PGIVTMNEYS VPKTLQAR F VKQIDFRPKD YLKHCLERSE FLLEKRIIQS GIALLLMLIW YRPVAEFAQS IQSRIVEWK ERLDKEFSLS VYQKMKFNVA MGIGVLDWLR NSDDDDEDSQ ENADKNEDGG EKNMEDSGHE TGIDSQSQG SFQAPQSSQS VHDHNQPYHI CRGFTCFKKP PTPPPEPET
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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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

Concentration0.5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average exposure time: 5.0 sec. / Average electron dose: 41.28 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: 2.6 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 130000
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 correctionType: NONE
Startup modelType of model: OTHER / Details: Abinitio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 80452
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT

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