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- EMDB-28195: SV40 T-Antigen helicase hexamer -

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Basic information

Entry
Database: EMDB / ID: EMD-28195
TitleSV40 T-Antigen helicase hexamer
Map data
Sample
  • Complex: SV40 T-Antigen
    • Protein or peptide: SV40 large antigen
KeywordsSV40 T-Antigen / VIRAL PROTEIN
Biological speciesBetapolyomavirus macacae
Methodsingle particle reconstruction / cryo EM / Resolution: 5.8 Å
AuthorsYuan Z / Langston L / Georgescu R / Li H / O'Donnell M
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM115809 United States
Howard Hughes Medical Institute (HHMI)M.E.O. United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding.
Authors: Lance D Langston / Zuanning Yuan / Roxana Georgescu / Huilin Li / Michael E O'Donnell /
Abstract: Duplication of DNA genomes requires unwinding of the double-strand (ds) DNA so that each single strand (ss) can be copied by a DNA polymerase. The genomes of eukaryotic cells are unwound by two ring- ...Duplication of DNA genomes requires unwinding of the double-strand (ds) DNA so that each single strand (ss) can be copied by a DNA polymerase. The genomes of eukaryotic cells are unwound by two ring-shaped hexameric helicases that initially encircle dsDNA but transition to ssDNA for function as replicative helicases. How the duplex is initially unwound, and the role of the two helicases in this process, is poorly understood. We recently described an initiation mechanism for eukaryotes in which the two helicases are directed inward toward one another and shear the duplex open by pulling on opposite strands of the duplex while encircling dsDNA [L. D. Langston, M. E. O'Donnell, , e46515 (2019)]. Two head-to-head T-Antigen helicases are long known to be loaded at the SV40 origin. We show here that T-Antigen tracks head (N-tier) first on ssDNA, opposite the direction proposed for decades. We also find that SV40 T-Antigen tracks directionally while encircling dsDNA and mainly tracks on one strand of the duplex in the same orientation as during ssDNA translocation. Further, two inward directed T-Antigen helicases on dsDNA are able to melt a 150-bp duplex. These findings explain the "rabbit ear" DNA loops observed at the SV40 origin by electron microscopy and reconfigure how the DNA loops emerge from the double hexamer relative to earlier models. Thus, the mechanism of DNA shearing by two opposing helicases is conserved in a eukaryotic viral helicase and may be widely used to initiate origin unwinding of dsDNA genomes.
History
DepositionSep 20, 2022-
Header (metadata) releaseNov 23, 2022-
Map releaseNov 23, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28195.png

Downloads & links

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Map

FileDownload / File: emd_28195.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.013
Minimum - Maximum-0.028409816 - 0.08780257
Average (Standard dev.)0.00037559983 (±0.0030752094)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 296.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_28195_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_28195_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SV40 T-Antigen

EntireName: SV40 T-Antigen
Components
  • Complex: SV40 T-Antigen
    • Protein or peptide: SV40 large antigen

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Supramolecule #1: SV40 T-Antigen

SupramoleculeName: SV40 T-Antigen / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Betapolyomavirus macacae

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Macromolecule #1: SV40 large antigen

MacromoleculeName: SV40 large antigen / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Betapolyomavirus macacae
SequenceString: MDKVLNREES LQLMDLLGLE RSAWGNIPLM RKAYLKKCKE FHPDKGGDEE KMKKMNTLYK KMEDGVKYAH QPDFGGFWDA TEIPTYGTDE WEQWWNAFNE ENLFCSEEMP SSDDEATADS QHSTPPKKKR KVEDPKDFPS ELLSFLSHAV FSNRTLACFA IYTTKEKAAL ...String:
MDKVLNREES LQLMDLLGLE RSAWGNIPLM RKAYLKKCKE FHPDKGGDEE KMKKMNTLYK KMEDGVKYAH QPDFGGFWDA TEIPTYGTDE WEQWWNAFNE ENLFCSEEMP SSDDEATADS QHSTPPKKKR KVEDPKDFPS ELLSFLSHAV FSNRTLACFA IYTTKEKAAL LYKKIMEKYS VTFISRHNSY NHNILFFLTP HRHRVSAINN YAQKLCTFSF LICKGVNKEY LMYSALTRDP FSVIEESLPG GLKEHDFNPE EAEETKQVSW KLVTEYAMET KCDDVLLLLG MYLEFQYSFE MCLKCIKKEQ PSHYKYHEKH YANAAIFADS KNQKTICQQA VDTVLAKKRV DSLQLTREQM LTNRFNDLLD RMDIMFGSTG SADIEEWMAG VAWLHCLLPK MDSVVYDFLK CMVYNIPKKR YWLFKGPIDS GKTTLAAALL ELCGGKALNV NLPLDRLNFE LGVAIDQFLV VFEDVKGTGG ESRDLPSGQG INNLDNLRDY LDGSVKVNLE KKHLNKRTQI FPPGIVTMNE FSVPKTLQAR FVKQIDFRAK DYLKHCLERS EFLLEKRIIQ SGIALLLMLI WYRPVAEFAQ SIQSRIVEWK ERLDKEFSLS VYQKMKFNVA MGIGVLDWLR NSDDDDEDSQ ENADKNEDGG EKNMEDSGHE TGIDSQSQGS FQAPQSSQSV HDHNQPYHIC RGFTCFKKPP TPPPEPET

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.0025 µm / Nominal defocus min: 0.0015 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1n25

Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 22375
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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