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- EMDB-3090: Structural basis for DNA strand separation by a hexameric replica... -

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Basic information

Entry
Database: EMDB / ID: EMD-3090
TitleStructural basis for DNA strand separation by a hexameric replicative helicase
Map dataReconstruction of full-length E1 helicase assembled on the DNA, labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA
Sample
  • Sample: Full-length E1 helicase-DNA complex labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA end of the DNA fork
  • Protein or peptide: Full-length hexameric E1 helicase
  • Protein or peptide: Monovalent tetrameric streptavidin
Keywordspapillomavirus / helicase / DNA replication fork / electron microscopy / structural analysis
Function / homology
Function and homology information


DNA 3'-5' helicase / biotin binding / DNA helicase activity / DNA replication / host cell nucleus / ATP hydrolysis activity / DNA binding / extracellular region / 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. / Avidin-like, conserved site / Avidin-like domain signature. / Avidin / : / Avidin/streptavidin / Avidin-like superfamily / Avidin family / Avidin-like domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Replication protein E1 / Streptavidin
Similarity search - Component
Biological speciesBovine papillomavirus / Streptomyces avidinii (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 18.0 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 12, 2015-
Map releaseAug 19, 2015-
UpdateJun 1, 2016-
Current statusJun 1, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.95
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.95
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3090.png

Downloads & links

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Map

FileDownload / File: emd_3090.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of full-length E1 helicase assembled on the DNA, labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.6 Å/pix.
x 200 pix.
= 320. Å		1.6 Å/pix.
x 200 pix.
= 320. Å		1.6 Å/pix.
x 200 pix.
= 320. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.6 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.95 / Movie #1: 0.95
Minimum - Maximum-7.55175066 - 16.889642720000001
Average (Standard dev.)0.07464474 (±0.85374743)
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
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-7.55216.8900.075

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Supplemental data

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Sample components

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Entire : Full-length E1 helicase-DNA complex labelled with monovalent tetr...

EntireName: Full-length E1 helicase-DNA complex labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA end of the DNA fork
Components
  • Sample: Full-length E1 helicase-DNA complex labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA end of the DNA fork
  • Protein or peptide: Full-length hexameric E1 helicase
  • Protein or peptide: Monovalent tetrameric streptavidin

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Supramolecule #1000: Full-length E1 helicase-DNA complex labelled with monovalent tetr...

SupramoleculeName: Full-length E1 helicase-DNA complex labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA end of the DNA fork
type: sample / ID: 1000
Oligomeric state: one MTS binds to one hexamer of full-length E1 helices via dsDNA
Number unique components: 3
Molecular weightExperimental: 460 KDa / Theoretical: 460 KDa / Method: Theoretical calculation

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Macromolecule #1: Full-length hexameric E1 helicase

MacromoleculeName: Full-length hexameric E1 helicase / type: protein_or_peptide / ID: 1 / Name.synonym: FLE1
Details: Full-length E1 helicase-DNA complex was labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA end of the DNA fork
Number of copies: 1 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Bovine papillomavirus / synonym: Bovine papillomavirus / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 410 KDa / Theoretical: 410 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET11c
SequenceUniProtKB: Replication protein E1

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Macromolecule #2: Monovalent tetrameric streptavidin

MacromoleculeName: Monovalent tetrameric streptavidin / type: protein_or_peptide / ID: 2 / Name.synonym: MTS / Number of copies: 1 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Streptomyces avidinii (bacteria) / synonym: Streptomyces avidinii
Molecular weightExperimental: 53 KDa / Theoretical: 53 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET21a
SequenceUniProtKB: Streptavidin

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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: Sample was stained with 2% uranyl acetate
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
TemperatureMin: 291 K / Max: 296 K / Average: 293 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateDec 14, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 1.6 µm / Number real images: 210 / Average electron dose: 20 e/Å2 / Camera length: 1000 / Details: No subframe averaging was used. / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 67000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 62000
Sample stageSpecimen holder: Negative stain holder / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticle picking was carried out automatically using BOXER software. Initial references were prepared using several manually selected protein complex images representing different views.
CTF correctionDetails: frames
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: OTHER / Software - Name: Imagic
Details: Two final maps were obtained using two different data set and using 750 classes for each
Number images used: 24000
Final angle assignmentDetails: Angular reconstitution
Final two d classificationNumber classes: 750

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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, Veda
DetailsThe domains were separately fitted by manual docking using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient
Output model


PDB Unreleased entry

PDB-5a9m:
Structural basis for DNA strand separation by a hexameric replicative helicase

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Atomic model buiding 2

Initial modelPDB ID:

1ksx


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking using Chimera based on the local correlation.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: local correlation coefficient
Output model


PDB Unreleased entry

PDB-5a9m:
Structural basis for DNA strand separation by a hexameric replicative helicase

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Atomic model buiding 3

Initial modelPDB ID:

3ry1


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D
SoftwareName: Chimera
DetailsThe tetramer structure was fitted in Chimera using the local correlation criterion.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient
Output model


PDB Unreleased entry

PDB-5a9m:
Structural basis for DNA strand separation by a hexameric replicative helicase

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