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- EMDB-11852: Bovine Papillomavirus E1 DNA helicase-replication fork complex -

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Basic information

Entry
Database: EMDB / ID: EMD-11852
TitleBovine Papillomavirus E1 DNA helicase-replication fork complex
Map data
Sample
  • Complex: BPV E1 DNA helicase-replication fork complex
    • Complex: DNA replication fork
      • DNA: DNA (40-MER)
      • DNA: DNA (36-MER)
    • Complex: Full-length E1 helicase
      • Protein or peptide: Replication protein E1
      • Protein or peptide: Replication protein E1
KeywordsDNA / virus / helicase / replisome / DNA replication. / DNA BINDING PROTEIN
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
Replication protein E1 / Replication protein E1
Similarity search - Component
Biological speciesBovine papillomavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsJaved A / Major B
Funding support United Kingdom, 2 items
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, 2020-
Header (metadata) releaseNov 17, 2021-
Map releaseNov 17, 2021-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7apd
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11852.png

Downloads & links

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Map

FileDownload / File: emd_11852.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 300 pix.
= 325.5 Å		1.09 Å/pix.
x 300 pix.
= 325.5 Å		1.09 Å/pix.
x 300 pix.
= 325.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.085 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0 / Movie #1: 4
Minimum - Maximum-27.11534 - 65.553160000000005
Average (Standard dev.)0.04843488 (±0.7824337)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 325.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0851.0851.085
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z325.500325.500325.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-27.11565.5530.048

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Supplemental data

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Sample components

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Entire : BPV E1 DNA helicase-replication fork complex

EntireName: BPV E1 DNA helicase-replication fork complex
Components
  • Complex: BPV E1 DNA helicase-replication fork complex
    • Complex: DNA replication fork
      • DNA: DNA (40-MER)
      • DNA: DNA (36-MER)
    • Complex: Full-length E1 helicase
      • Protein or peptide: Replication protein E1
      • Protein or peptide: Replication protein E1

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Supramolecule #1: BPV E1 DNA helicase-replication fork complex

SupramoleculeName: BPV E1 DNA helicase-replication fork complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 413.4 KDa

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Supramolecule #2: DNA replication fork

SupramoleculeName: DNA replication fork / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3-#4
Details: Consists of two strands and three regions: dsDNA, 5'ssDNA lagging strand and 3' ssDNA leading strand.
Source (natural)Organism: Bovine papillomavirus

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Supramolecule #3: Full-length E1 helicase

SupramoleculeName: Full-length E1 helicase / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Details: Composed 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).
Source (natural)Organism: Bovine papillomavirus

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Macromolecule #1: Replication protein E1

MacromoleculeName: Replication protein E1 / type: protein_or_peptide / ID: 1 / Details: OBD domains from subunits B and E. / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Bovine papillomavirus
Molecular weightTheoretical: 17.162084 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSRATVFKLG LFKSLFLCSF HDITRLFKND KTTNQQWVLA VFGLAEVFFE ASFELLKKQC SFLQMQKRSH EGGTCAVYLI CFNTAKSRE TVRNLMANML NVREECLMLQ PPKIRGLSAA LFWFKSSLSP ATLKHGALPE WIRAQTTLNA AAA

UniProtKB: Replication protein E1

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Macromolecule #2: Replication protein E1

MacromoleculeName: Replication protein E1 / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Bovine papillomavirus
Molecular weightTheoretical: 33.859172 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TEKFDFGTMV QWAYDHKYAE ESKIAYEYAL AAGSDSNARA FLATNSQAKH VKDCATMVRH YLRAETQALS MPAYIKARCK LATGEGSWK SILTFFNYQN IELITFINAL KLWLKGIPKK NCLAFIGPPN TGKSMLCNSL IHFLGGSVLS FANHKSHFWL A SLADTRAA ...String:
TEKFDFGTMV QWAYDHKYAE ESKIAYEYAL AAGSDSNARA FLATNSQAKH VKDCATMVRH YLRAETQALS MPAYIKARCK LATGEGSWK SILTFFNYQN IELITFINAL KLWLKGIPKK NCLAFIGPPN TGKSMLCNSL IHFLGGSVLS FANHKSHFWL A SLADTRAA LVDDATHACW RYFDTYLRNA LDGYPVSIDR KHKAAVQIKA PPLLVTSNID VQAEDRYLYL HSRVQTFRFE QP CTDESGE QPFNITDADW KSFFVRLWGR LDLIDEEEDS EEDGDSMRTF TCSARNTNAV D

UniProtKB: Replication protein E1

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Macromolecule #3: DNA (40-MER)

MacromoleculeName: DNA (40-MER) / type: dna / ID: 3 / Details: 5'-3' ssDNA strand of the DNA replication fork. / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bovine papillomavirus
Molecular weightTheoretical: 12.142779 KDa
SequenceString:
(DT)(DG)(DT)(DA)(DT)(DT)(DT)(DC)(DA)(DC) (DA)(DC)(DC)(DG)(DC)(DA)(DC)(DC)(DT)(DC) (DA)(DG)(DC)(DG)(DC)(DG)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)

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Macromolecule #4: DNA (36-MER)

MacromoleculeName: DNA (36-MER) / type: dna / ID: 4 / Details: 3'-5' ssDNA strand of the DNA replication fork. / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bovine papillomavirus
Molecular weightTheoretical: 11.08009 KDa
SequenceString:
(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DG)(DT)(DG) (DC)(DG)(DC)(DG)(DC)(DT)(DG)(DA)(DG)(DG) (DT)(DG)(DC)(DG)(DG)(DT)(DG)(DT)(DG) (DA)(DA)(DA)(DT)(DA)(DC)(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

Concentration0.05 mg/mL
BufferpH: 7.2
Component:
ConcentrationName
20.0 mMHepes
200.0 mMNaCl
1.0 mMEDTA
0.1 mMPMSF
2.0 mMDTT
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 3 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Details: 3 ul of sample was applied Lacey ultra-thin carbon film grids..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 95.0 K / Max: 98.0 K
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 12136 / Average exposure time: 3.0 sec. / Average electron dose: 50.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 47170 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000
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

Particle selectionNumber selected: 568120
Startup modelType of model: OTHER
Details: Cryo-EM 3D reconstruction of E1-Helicase using IMAGIC-5 software, in-house.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.9) / Number images used: 81831
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9)
Final 3D classificationNumber classes: 6 / Avg.num./class: 15000 / Software - Name: cryoSPARC (ver. 2.9)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

2gxa

source_name: PDB, initial_model_type: experimental model

1ksy

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 102 / Target criteria: Correlation coefficient
Output model

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

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