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- EMDB-50002: SV40 large T antigen assembly with DNA in presence of ADP -

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Basic information

Entry
Database: EMDB / ID: EMD-50002
TitleSV40 large T antigen assembly with DNA in presence of ADP
Map dataPost-processed map (EMReady).
Sample
  • Complex: Large T antigen with DNA in presence of ADP.
    • Complex: Large T antigen
      • Protein or peptide: Large T antigen
    • Complex: DNA
      • DNA: DNA
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
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 / helicase activity / isomerase 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 / helicase activity / isomerase 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: 3.38 Å
AuthorsShahid T
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
DepositionMar 29, 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_50002.png

Downloads & links

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Map

FileDownload / File: emd_50002.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed map (EMReady).
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 320 pix.
= 320. Å		1 Å/pix.
x 320 pix.
= 320. Å		1 Å/pix.
x 320 pix.
= 320. Å

Surface

Projections

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Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.637
Minimum - Maximum-0.636001 - 18.007048000000001
Average (Standard dev.)-0.009545609 (±0.4972727)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50002_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_50002_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_50002_half_map_2.map
Projections & Slices
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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
      • Protein or peptide: Large T antigen
    • Complex: DNA
      • DNA: DNA
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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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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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: 81.732953 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDKVLNREES LQLMDLLGLE RSAWGNIPLM RKAYLKKCKE FHPDKGGDEE KMKKMNTLYK KMEDGVKYAH QPDFGGFWDA TEIPTYGTD EWEQWWNAFN EENLFCSEEM PSSDDEATAD SQHSTPPKKK RKVEDPKDFP SELLSFLSHA VFSNRTLACF A IYTTKEKA ...String:
MDKVLNREES LQLMDLLGLE RSAWGNIPLM RKAYLKKCKE FHPDKGGDEE KMKKMNTLYK KMEDGVKYAH QPDFGGFWDA TEIPTYGTD EWEQWWNAFN EENLFCSEEM PSSDDEATAD SQHSTPPKKK RKVEDPKDFP SELLSFLSHA VFSNRTLACF A IYTTKEKA ALLYKKIMEK YSVTFISRHN SYNHNILFFL TPHRHRVSAI NNYAQKLCTF SFLICKGVNK EYLMYSALTR DP FSVIEES LPGGLKEHDF NPEEAEETKQ VSWKLVTEYA METKCDDVLL LLGMYLEFQY SFEMCLKCIK KEQPSHYKYH EKH YANAAI FADSKNQKTI CQQAVDTVLA KKRVDSLQLT REQMLTNRFN DLLDRMDIMF GSTGSADIEE WMAGVAWLHC LLPK MDSVV YDFLKCMVYN IPKKRYWLFK GPIDSGKTTL AAALLELCGG KALNVNLPLD RLNFELGVAI DQFLVVFEDV KGTGG ESRD LPSGQGINNL DNLRDYLDGS VKVNLEKKHL NKRTQIFPPG IVTMNEYSVP KTLQARFVKQ IDFRPKDYLK HCLERS EFL LEKRIIQSGI ALLLMLIWYR PVAEFAQSIQ SRIVEWKERL DKEFSLSVYQ KMKFNVAMGI GVLDWLRNSD DDDEDSQ EN ADKNEDGGEK NMEDSGHETG IDSQSQGSFQ APQSSQSVHD HNQPYHICRG FTCFKKPPTP PPEPET

UniProtKB: Large T antigen

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Macromolecule #2: DNA

MacromoleculeName: DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.084392 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, 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 ADP.
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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.38 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
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1svm

source_name: PDB, initial_model_type: experimental model

2gxa

chain_id: M, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-9evh:
SV40 large T antigen assembly with DNA in presence of ADP

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