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- EMDB-3087: Structural basis for DNA strand separation by a hexameric replica... -

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Basic information

Entry
Database: EMDB / ID: EMD-3087
TitleStructural basis for DNA strand separation by a hexameric replicative helicase
Map dataA hexamer of the helicase domain (E1HD) of the full length E1 helicase (E1FL)
Sample
  • Sample: Helicase domain of the hexameric full-length E1 helicase-DNA complex
  • Protein or peptide: Helicase domain (301-605 aa)
Keywordspapillomavirus / helicase / DNA replication fork / electron microscopy / structural analysis
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
Similarity search - Component
Biological speciesBovine papillomavirus
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 Å
AuthorsChaban Y / Stead JA / Ryzhenkova K / Whelan F / Lamber K / Antson A / Sanders CM / Orlova EV
CitationJournal: Nucleic Acids Res / Year: 2015
Title: Structural basis for DNA strand separation by a hexameric replicative helicase.
Authors: Yuriy Chaban / Jonathan A Stead / Ksenia Ryzhenkova / Fiona Whelan / Ekaterina P Lamber / Alfred Antson / Cyril M Sanders / Elena V Orlova /
Abstract: Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined ...Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5' ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted 'steric exclusion' model for dsDNA unwinding, the active 3' ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5' passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.
History
DepositionJul 9, 2015-
Header (metadata) releaseAug 5, 2015-
Map releaseAug 19, 2015-
UpdateOct 7, 2015-
Current statusOct 7, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a9k
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image3087.png

Downloads & links

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Map

FileDownload / File: emd_3087.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationA hexamer of the helicase domain (E1HD) of the full length E1 helicase (E1FL)
Voxel sizeX=Y=Z: 1.6 Å
Density
Contour LevelBy AUTHOR: 0.011 / Movie #1: 0.011
Minimum - Maximum-0.16802455 - 0.43106768
Average (Standard dev.)0.00074986 (±0.0126535)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.61.61.6
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-0.1680.4310.001

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Supplemental data

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Sample components

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Entire : Helicase domain of the hexameric full-length E1 helicase-DNA complex

EntireName: Helicase domain of the hexameric full-length E1 helicase-DNA complex
Components
  • Sample: Helicase domain of the hexameric full-length E1 helicase-DNA complex
  • Protein or peptide: Helicase domain (301-605 aa)

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Supramolecule #1000: Helicase domain of the hexameric full-length E1 helicase-DNA complex

SupramoleculeName: Helicase domain of the hexameric full-length E1 helicase-DNA complex
type: sample / ID: 1000 / Oligomeric state: one hexamer / Number unique components: 1
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa / Method: Theoretical calculation

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Macromolecule #1: Helicase domain (301-605 aa)

MacromoleculeName: Helicase domain (301-605 aa) / type: protein_or_peptide / ID: 1 / Name.synonym: E1HD / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Bovine papillomavirus / synonym: bovine papillomavirus / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET11c
SequenceUniProtKB: Replication protein E1

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3 mg/mL
BufferpH: 8
Details: 10 mM Tris-Cl pH 8.0, 225 mM NaCl, 2 mM DTT, 0.1 mM PMSF, 0.1 mM EDTA
StainingType: NEGATIVE
Details: Grids were stained with 2% uranyl acetate for 1 minute
GridDetails: Sample was applied on to carbon-coated copper grids (400 mesh, freshly glow-discharged in air)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 67000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 62000
Sample stageSpecimen holder: Negative staining holder / Specimen holder model: OTHER
TemperatureMin: 291 K / Max: 296 K / Average: 293 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateFeb 18, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 1.6 µm / Number real images: 30 / Average electron dose: 20 e/Å2 / Camera length: 1000 / Details: No subframe averaging was used. / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: frames,
Final two d classificationNumber classes: 450
Final angle assignmentDetails: Angular reconstitution, 6-fold symmetry
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: OTHER / Software - Name: Imagic, CTFit, CTFFIND3 / Details: Final map was calculated from 450 classes / Number images used: 8265
DetailsParticle picking was carried out automatically using BOXER software. Initial references were prepared using several manually selected protein complex images representing different views.

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Atomic model buiding 1

Initial modelPDB ID:

2v9p


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F
SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-5a9k:
Structural basis for DNA strand separation by a hexameric replicative helicase

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