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- EMDB-1022: Three-dimensional reconstructions from cryoelectron microscopy im... -

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Basic information

Entry
Database: EMDB / ID: EMD-1022
TitleThree-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC.
Map dataDnaB
Sample
  • Sample: DnaB from E.coli
  • Protein or peptide: DnaB helicase
Function / homology: / DNA helicase activity
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 34.5 Å
AuthorsSan Martin C
CitationJournal: Structure / Year: 1998
Title: Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC.
Authors: C San Martin / M Radermacher / B Wolpensinger / A Engel / C S Miles / N E Dixon / J M Carazo /
Abstract: BACKGROUND: DNA helicases play a fundamental role in all aspects of nucleic acid metabolism and defects in these enzymes have been implicated in a number of inherited human disorders. DnaB is the ...BACKGROUND: DNA helicases play a fundamental role in all aspects of nucleic acid metabolism and defects in these enzymes have been implicated in a number of inherited human disorders. DnaB is the major replicative DNA helicase in Escherichia coli and has been used as a model system for studying the structure and function of hexameric helicases. The native protein is a hexamer of identical subunits, which in solution forms a complex with six molecules of the loading protein DnaC. DnaB is delivered from this complex onto the DNA template, with the subsequent release of DnaC. We report here the structures of the DnaB helicase hexamer and its complex with DnaC under a defined set of experimental conditions, as determined by three-dimensional cryoelectron microscopy. It was hoped that the structures would provide insight into the mechanisms of helicase activity.
RESULTS: The DnaB structure reveals that six DnaB monomers assemble as three asymmetric dimers to form a polar, ring-like hexamer. The hexamer has two faces, one displaying threefold and the other ...RESULTS: The DnaB structure reveals that six DnaB monomers assemble as three asymmetric dimers to form a polar, ring-like hexamer. The hexamer has two faces, one displaying threefold and the other sixfold symmetry. The six DnaC protomers bind tightly to the sixfold face of the DnaB hexamer. This is the first report of a three-dimensional structure of a helicase obtained using cryoelectron microscopy, and the first report of the structure of a helicase in complex with a loading protein.
CONCLUSIONS: The structures of the DnaB helicase and its complex with DnaC reveal some interesting structural features relevant to helicase function and to the assembly of the two-protein complex. ...CONCLUSIONS: The structures of the DnaB helicase and its complex with DnaC reveal some interesting structural features relevant to helicase function and to the assembly of the two-protein complex. The results presented here provide a basis for a more complete understanding of the structure and function of these important proteins.
History
DepositionFeb 10, 2003-
Header (metadata) releaseFeb 12, 2003-
Map releaseFeb 12, 2003-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6.678072
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 6.678072
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1022.map.gz / Format: CCP4 / Size: 478.5 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDnaB
Voxel sizeX=Y=Z: 3.8 Å
Density
Contour Level1: 6.98 / Movie #1: 6.678072
Minimum - Maximum-9.14113 - 11.271599999999999
Average (Standard dev.)0.0000960484 (±2.93696)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions505050
Spacing505050
CellA=B=C: 190 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.83.83.8
M x/y/z505050
origin x/y/z0.0000.0000.000
length x/y/z190.000190.000190.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS505050
D min/max/mean-9.14111.2720.000

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Supplemental data

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Sample components

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Entire : DnaB from E.coli

EntireName: DnaB from E.coli
Components
  • Sample: DnaB from E.coli
  • Protein or peptide: DnaB helicase

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Supramolecule #1000: DnaB from E.coli

SupramoleculeName: DnaB from E.coli / type: sample / ID: 1000 / Oligomeric state: homohexamer / Number unique components: 1
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: DnaB helicase

MacromoleculeName: DnaB helicase / type: protein_or_peptide / ID: 1 / Name.synonym: DnaB / Number of copies: 6 / Oligomeric state: homohexamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: AN1459/pPS560 / synonym: E. coli
Molecular weightTheoretical: 300 KDa
Recombinant expressionOrganism: AN1459/pPS560 / Recombinant plasmid: pPS560
SequenceGO: DNA helicase activity / InterPro: INTERPRO: IPR001198

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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

Concentration.04 mg/mL
BufferpH: 7.6
Details: 50 mM Tris.HCl pH 7.6 2 mM DTT 5 mM MgCl2 200 mM NaCl 0.25 mM ADP
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: double side blotting device
Method: samples were adsorbed onto carbon-coated molybdenum holey grids after 30s glow discharge, and vitrified by quick plunging in liquid ethane in a double-side blotting device

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Electron microscopy

MicroscopeFEI/PHILIPS EM420
Electron beamAcceleration voltage: 100 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Gatan626 / Specimen holder model: GATAN LIQUID NITROGEN / Tilt angle max: 45
TemperatureAverage: 98 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: EIKONIX IEEE 488 / Number real images: 27 / Average electron dose: 10 e/Å2 / Bits/pixel: 8

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Image processing

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 34.5 Å / Resolution method: OTHER / Software - Name: Xmipp, SPIDER / Number images used: 1369

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