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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 data
SampleDnaB from E.coli:
DnaB helicase
Function / homologyin:IPR001198: / 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 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. The results presented here provide a basis for a more complete understanding of the structure and function of these important proteins.
DateDeposition: Feb 10, 2003 / Header (metadata) release: Feb 12, 2003 / Map release: Feb 12, 2003 / Update: May 26, 2011

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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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.8 Å/pix.
x 50 pix.
= 190. Å
3.8 Å/pix.
x 50 pix.
= 190. Å
3.8 Å/pix.
x 50 pix.
= 190. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.8 Å
Density
Contour LevelPrimary: 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 / Oligomeric State: homohexamer / Number of components: 1
MassTheoretical: 300 kDa

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Component #1: protein, DnaB helicase

ProteinName: DnaB helicase / a.k.a: DnaB / Oligomeric Details: homohexamer / Recombinant expression: Yes / Number of Copies: 6
MassTheoretical: 300 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: AN1459/pPS560
Source (engineered)Expression System: AN1459/pPS560 / Vector: pPS560
External referencesInterPro: InterPro: IPR001198 / Gene Ontology: DNA helicase activity

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.04 mg/mL
Buffer solution: 50 mM Tris.HCl pH 7.6 2 mM DTT 5 mM MgCl2 200 mM NaCl 0.25 mM ADP
pH: 7.6
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
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
Details: Vitrification instrument: double side blotting device

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Electron microscopy imaging

ImagingMicroscope: FEI/PHILIPS EM420
Electron gunElectron source: LAB6 / Accelerating voltage: 100 kV / Electron dose: 10 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 60000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 2000 nm
Specimen HolderHolder: Gatan626 / Model: GATAN LIQUID NITROGEN / Tilt Angle: - 45 ° / Temperature: 98
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 27 / Scanner: EIKONIX IEEE 488 / Bit depth: 8

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 1369 / Applied symmetry: C3 (3 fold cyclic)
3D reconstructionAlgorithm: Random conical tilt / Software: Xmipp, SPIDER / Resolution: 34.5 Å / Resolution method: DPR at 45 degrees

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