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- EMDB-50010: SV40 LTAg assembly with DNA in presence of AMPPNP and Mg2+. -

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Basic information

Entry
Database: EMDB / ID: EMD-50010
TitleSV40 LTAg assembly with DNA in presence of AMPPNP and Mg2+.
Map dataEMReady2 post-processed map.
Sample
  • Complex: SV40 large T antigen assembly with DNA in presence of AMPPNP and Mg2+.
    • Complex: SV40 large T antigen assembly
      • Protein or peptide: Large T antigen
    • Complex: DNA
      • DNA: DNA
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
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 ...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
Biological speciesBetapolyomavirus macacae / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsShahid T / De Biasio A / Clark A / Savva CG
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
DepositionApr 1, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50010.png

Downloads & links

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Map

FileDownload / File: emd_50010.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEMReady2 post-processed map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 256 pix.
= 238.08 Å		0.93 Å/pix.
x 256 pix.
= 238.08 Å		0.93 Å/pix.
x 256 pix.
= 238.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.88
Minimum - Maximum-0.132362 - 22.115738
Average (Standard dev.)0.07564403 (±0.7813242)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 238.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_50010_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_50010_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SV40 large T antigen assembly with DNA in presence of AMPPNP and Mg2+.

EntireName: SV40 large T antigen assembly with DNA in presence of AMPPNP and Mg2+.
Components
  • Complex: SV40 large T antigen assembly with DNA in presence of AMPPNP and Mg2+.
    • Complex: SV40 large T antigen assembly
      • Protein or peptide: Large T antigen
    • Complex: DNA
      • DNA: DNA
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: SV40 large T antigen assembly with DNA in presence of AMPPNP and Mg2+.

SupramoleculeName: SV40 large T antigen assembly with DNA in presence of AMPPNP and Mg2+.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: SV40 large T antigen assembly

SupramoleculeName: SV40 large T antigen assembly / 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: 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: 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: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 6 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration1 mg/mL
BufferpH: 7.5
Details: 50 mM TrisHCl (pH 7.5), 1 mM DTT, 100 mM NaCl, 5 mM MgCl, 3 mM AMP-PMP.
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Details: 40 mA.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 6127 / Average electron dose: 38.7 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: 1.5 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

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number images used: 235707
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Residue range: 266-627 / Chain - Source name: Other / Chain - Initial model type: experimental model / Details: LTAg-ATP-FlapDNA structure (associated deposition)
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-9evp:
SV40 LTAg assembly with DNA in presence of AMPPNP and Mg2+.

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