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- EMDB-3091: Structural basis for DNA strand separation by a hexameric replica... -

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Basic information

Entry
Database: EMDB / ID: EMD-3091
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 and with FAB on the 5' ssDNA
Sample
  • Sample: Full-length E1 helicase-DNA complex labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA end and with FAB on the 5' ssDNA end
  • Protein or peptide: Monovalent tetrameric streptavidin
  • Protein or peptide: Full-length hexametric E1 helicase complex
  • Protein or peptide: IGG2A-KAPPA 26-10 FAB
Keywordspapillomavirus / helicase / DNA replication fork / electron microscopy / structural analysis
Function / homology
Function and homology information


biotin binding / DNA helicase activity / DNA helicase / 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 speciesStreptomyces avidinii (bacteria) / Bovine papillomavirus / Mus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 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-
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: 0.0034
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0034
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image3091.png

Downloads & links

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Map

FileDownload / File: emd_3091.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 and with FAB on the 5' ssDNA
Voxel sizeX=Y=Z: 1.6 Å
Density
Contour LevelBy AUTHOR: 0.0034 / Movie #1: 0.0034
Minimum - Maximum-0.01647516 - 0.03098119
Average (Standard dev.)0.00018996 (±0.00157235)
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-0.0160.0310.000

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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 and with FAB on the 5' ssDNA end
Components
  • Sample: Full-length E1 helicase-DNA complex labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA end and with FAB on the 5' ssDNA end
  • Protein or peptide: Monovalent tetrameric streptavidin
  • Protein or peptide: Full-length hexametric E1 helicase complex
  • Protein or peptide: IGG2A-KAPPA 26-10 FAB

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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 and with FAB on the 5' ssDNA end
type: sample / ID: 1000
Oligomeric state: one MTS and one FAB are bound to one hexameter of full-length E1 helicase via DNA fork
Number unique components: 4
Molecular weightExperimental: 490 KDa / Theoretical: 490 KDa / Method: Theoretical calculation

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Macromolecule #1: Monovalent tetrameric streptavidin

MacromoleculeName: Monovalent tetrameric streptavidin / type: protein_or_peptide / ID: 1 / Name.synonym: MTS
Details: Monovalent tetrameric streptavidin (MTS) was bound to the dsDNA via biotin
Number of copies: 1 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Streptomyces avidinii (bacteria) / synonym: Streptomyces avidinii
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET21a
SequenceUniProtKB: Streptavidin

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Macromolecule #2: Full-length hexametric E1 helicase complex

MacromoleculeName: Full-length hexametric E1 helicase complex / type: protein_or_peptide / ID: 2 / Name.synonym: FLE1
Details: Full-length E1 helicase-DNA complex was labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA and with FAB on the 5' ssDNA
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 (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pet11c
SequenceUniProtKB: Replication protein E1

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Macromolecule #3: IGG2A-KAPPA 26-10 FAB

MacromoleculeName: IGG2A-KAPPA 26-10 FAB / type: protein_or_peptide / ID: 3 / Name.synonym: FAB
Details: Full-length E1 helicase-DNA complex was labelled with monovalent tetrameric streptavidin (MTS) on the dsDNA and with FAB on the 5' ssDNA
Number of copies: 1 / Oligomeric state: hetero dimer / Recombinant expression: No
Source (natural)Organism: Mus musculus (house mouse) / synonym: House mouse
Molecular weightExperimental: 30 KDa / Theoretical: 30 KDa

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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
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 stain 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
DateJun 5, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 1.6 µm / Number real images: 260 / 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: 750
Final angle assignmentDetails: Angular reconstitution
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: Imagic / Details: Final map was calculated from 750 classes / Number images used: 18000
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:

1igj


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
DetailsThe entire FAB molecule was fitted by manual docking using Chimera using the local correlation criteria
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: local correlation coefficient

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Atomic model buiding 2

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 MTS was fitted by manual docking in Chimera using the local correlation
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: local correlation coefficient

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Atomic model buiding 3

Initial modelPDB ID:

1ksx


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

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Atomic model buiding 4

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
DetailsRigid body fitting of the hexamer E1HD into the map
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient

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