|Entry||Database: EMDB / ID: EMD-1022|
|Title||Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC.|
|Sample||DnaB from E.coli:|
|Function / homology||in:IPR001198: / DNA helicase activity|
Function and homology information
|Biological species||Escherichia coli (E. coli)|
|Method||single particle reconstruction / cryo EM / Resolution: 34.5 Å|
|Authors||San Martin C|
|Citation||Journal: 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.
|Date||Deposition: Feb 10, 2003 / Header (metadata) release: Feb 12, 2003 / Map release: Feb 12, 2003 / Update: May 26, 2011|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_1022.map.gz / Format: CCP4 / Size: 478.5 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 3.8 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire DnaB from E.coli
|Entire||Name: DnaB from E.coli / Oligomeric State: homohexamer / Number of components: 1|
|Mass||Theoretical: 300 kDa|
-Component #1: protein, DnaB helicase
|Protein||Name: DnaB helicase / a.k.a: DnaB / Oligomeric Details: homohexamer / Recombinant expression: Yes / Number of Copies: 6|
|Mass||Theoretical: 300 kDa|
|Source||Species: Escherichia coli (E. coli) / Strain: AN1459/pPS560|
|Source (engineered)||Expression System: AN1459/pPS560 / Vector: pPS560|
|External references||InterPro: InterPro: IPR001198 / Gene Ontology: DNA helicase activity|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen 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
|Vitrification||Instrument: 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
-Electron microscopy imaging
|Imaging||Microscope: FEI/PHILIPS EM420|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 100 kV / Electron dose: 10 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 60000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 2000 nm|
|Specimen Holder||Holder: Gatan626 / Model: GATAN LIQUID NITROGEN / Tilt Angle: - 45 ° / Temperature: 98|
|Camera||Detector: KODAK SO-163 FILM|
|Image acquisition||Number of digital images: 27 / Scanner: EIKONIX IEEE 488 / Bit depth: 8|
|Processing||Method: single particle reconstruction / Number of projections: 1369 / Applied symmetry: C3 (3 fold cyclic)|
|3D reconstruction||Algorithm: Random conical tilt / Software: Xmipp, SPIDER / Resolution: 34.5 Å / Resolution method: DPR at 45 degrees|
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