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- PDB-7apd: Bovine Papillomavirus E1 DNA helicase-replication fork complex -

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Basic information

Entry
Database: PDB / ID: 7apd
TitleBovine Papillomavirus E1 DNA helicase-replication fork complex
Components
  • (Replication protein E1) x 2
  • DNA (36-MER)
  • DNA (40-MER)
KeywordsDNA BINDING PROTEIN / DNA / virus / helicase / replisome / DNA replication.
Function / homology
Function and homology information


DNA helicase activity / DNA helicase / DNA replication / host cell nucleus / ATP hydrolysis activity / DNA binding / ATP binding
Similarity search - Function
DNA helicase E1, C-terminal, Papillomavirus / DNA helicase E1, N-terminal, Papillomavirus / Replication protein E1, papillomavirus / DNA helicase E1, DNA-binding domain, papillomavirus / DNA helicase E1, DNA-binding domain superfamily, papillomavirus / Papillomavirus helicase / E1 Protein, N terminal domain / Papillomavirus E1, DNA-binding domain / Zinc finger, large T-antigen D1 domain superfamily / Helicase, superfamily 3, DNA virus ...DNA helicase E1, C-terminal, Papillomavirus / DNA helicase E1, N-terminal, Papillomavirus / Replication protein E1, papillomavirus / DNA helicase E1, DNA-binding domain, papillomavirus / DNA helicase E1, DNA-binding domain superfamily, papillomavirus / Papillomavirus helicase / E1 Protein, N terminal domain / Papillomavirus E1, DNA-binding domain / Zinc finger, large T-antigen D1 domain superfamily / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / Replication protein E1 / Replication protein E1
Similarity search - Component
Biological speciesBovine papillomavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsJaved, A. / Major, B. / Stead, J. / Sanders, C.M. / Orlova, E.V.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R002622/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R001685/1 United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Unwinding of a DNA replication fork by a hexameric viral helicase.
Authors: Abid Javed / Balazs Major / Jonathan A Stead / Cyril M Sanders / Elena V Orlova /
Abstract: Hexameric helicases are motor proteins that unwind double-stranded DNA (dsDNA) during DNA replication but how they are optimised for strand separation is unclear. Here we present the cryo-EM ...Hexameric helicases are motor proteins that unwind double-stranded DNA (dsDNA) during DNA replication but how they are optimised for strand separation is unclear. Here we present the cryo-EM structure of the full-length E1 helicase from papillomavirus, revealing all arms of a bound DNA replication fork and their interactions with the helicase. The replication fork junction is located at the entrance to the helicase collar ring, that sits above the AAA + motor assembly. dsDNA is escorted to and the 5´ single-stranded DNA (ssDNA) away from the unwinding point by the E1 dsDNA origin binding domains. The 3´ ssDNA interacts with six spirally-arranged β-hairpins and their cyclical top-to-bottom movement pulls the ssDNA through the helicase. Pulling of the RF against the collar ring separates the base-pairs, while modelling of the conformational cycle suggest an accompanying movement of the collar ring has an auxiliary role, helping to make efficient use of ATP in duplex unwinding.
History
DepositionOct 16, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-11852
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
G: Replication protein E1
B: Replication protein E1
C: Replication protein E1
D: Replication protein E1
E: Replication protein E1
F: Replication protein E1
A: Replication protein E1
H: Replication protein E1
P: DNA (40-MER)
T: DNA (36-MER)


Theoretical massNumber of molelcules
Total (without water)260,70210
Polymers260,70210
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Replication protein E1 / ATP-dependent helicase E1


Mass: 17162.084 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: OBD domains from subunits B and E. / Source: (gene. exp.) Bovine papillomavirus / Gene: E1 / Production host: Escherichia coli (E. coli) / References: UniProt: C5IAS0, DNA helicase
#2: Protein
Replication protein E1 / ATP-dependent helicase E1


Mass: 33859.172 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bovine papillomavirus / Gene: E1 / Production host: Escherichia coli (E. coli) / References: UniProt: P03116, DNA helicase
#3: DNA chain DNA (40-MER)


Mass: 12142.779 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: 5'-3' ssDNA strand of the DNA replication fork. / Source: (synth.) Bovine papillomavirus
#4: DNA chain DNA (36-MER)


Mass: 11080.090 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: 3'-5' ssDNA strand of the DNA replication fork. / Source: (synth.) Bovine papillomavirus

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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-IDSourceDetails
1BPV E1 DNA helicase-replication fork complexCOMPLEXall0MULTIPLE SOURCES
2DNA replication forkCOMPLEX#3-#41RECOMBINANTConsists of two strands and three regions: dsDNA, 5'ssDNA lagging strand and 3' ssDNA leading strand.
3Full-length E1 helicaseCOMPLEX#1-#21RECOMBINANTComposed of six subunits; Two subunits contain the Origin Binding domains (Chains G, H), all six subunits contain the helicase domain and the C-terminal tail (Chains A-F).
Molecular weightValue: 0.4134 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Bovine papillomavirus10571
33Bovine papillomavirus10571
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Synthetic construct (others)32630
33Escherichia col562
Buffer solutionpH: 7.2
Buffer component
IDConc.NameBuffer-ID
120 mMHepes1
2200 mMNaCl1
31 mMEDTA1
40.1 mMPMSF1
52 mMDTT1
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: 3 ul of sample was applied Lacey ultra-thin carbon film grids.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 47170 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 98 K / Temperature (min): 95 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 50.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 12136
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5.2 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fittingRigid-body
8Cootmodel fittingReal-space
9iMODFITmodel fittingFlexible fit
11cryoSPARC2.9initial Euler assignment
12cryoSPARC2.9final Euler assignment
13cryoSPARC2.9classification
14cryoSPARC2.93D reconstruction
21Cootmodel refinementReal-space
22PHENIXmodel refinementReal-space
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 568120
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81831 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 102 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
12GXA

2gxa

12GXA1PDBexperimental model
21KSY

1ksy

11KSY2PDBexperimental model

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