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- EMDB-1225: Loading a ring: structure of the Bacillus subtilis DnaB protein, ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1225
TitleLoading a ring: structure of the Bacillus subtilis DnaB protein, a co-loader of the replicative helicase.
Map dataBacillus subtilis DnaB
Sample
  • Sample: Bacillus subtilis DnaB
  • Protein or peptide: DnaB
Function / homology:
Function and homology information
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsNunez-Ramirez R / Velten M / Rivas G / Polard P / Carazo JM / Donate LE
CitationJournal: J Mol Biol / Year: 2007
Title: Loading a ring: structure of the Bacillus subtilis DnaB protein, a co-loader of the replicative helicase.
Authors: Rafael Núñez-Ramírez / Marion Velten / Germán Rivas / Patrice Polard / José María Carazo / Luis Enrique Donate /
Abstract: Loading of the ring-shaped replicative helicase is a critical step in the initiation of DNA replication. Bacillus subtilis has adopted a two-protein strategy to load its hexameric replicative ...Loading of the ring-shaped replicative helicase is a critical step in the initiation of DNA replication. Bacillus subtilis has adopted a two-protein strategy to load its hexameric replicative helicase: DnaB and DnaI interact with the helicase and mediate its delivery onto DNA. We present here the 3D electron microscopy structure of the DnaB protein, along with a detailed analysis of both its oligomeric state and its domain organization. DnaB is organized as an asymmetric tetramer that is comprised of two stacked components, one arranged as a closed collar and the other as an open sigma shape. Intriguingly, the 3D map of DnaB exhibits an overall architecture similar to the structure of the Escherichia coli gamma-complex, the loader of the ring-shaped processivity factor. We propose a model whereby each DnaB monomer participates in both stacked components of the tetramer and displays a different overall shape. This asymmetric quaternary organization could be a general feature of ring loaders.
History
DepositionApr 25, 2006-
Header (metadata) releaseMay 16, 2006-
Map releaseMay 16, 2007-
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: 4.304519715
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.304519715
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1225.gif

Downloads & links

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Map

FileDownload / File: emd_1225.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBacillus subtilis DnaB
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4 Å/pix.
x 64 pix.
= 256. Å		4 Å/pix.
x 64 pix.
= 256. Å		4 Å/pix.
x 64 pix.
= 256. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.24 / Movie #1: 4.3045197
Minimum - Maximum-4.78986 - 8.93615
Average (Standard dev.)-0.0355513 (±0.939863)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin111
Dimensions646464
Spacing646464
CellA=B=C: 256 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z444
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z256.000256.000256.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS111
NC/NR/NS646464
D min/max/mean-4.7908.936-0.036

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Supplemental data

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Sample components

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Entire : Bacillus subtilis DnaB

EntireName: Bacillus subtilis DnaB
Components
  • Sample: Bacillus subtilis DnaB
  • Protein or peptide: DnaB

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Supramolecule #1000: Bacillus subtilis DnaB

SupramoleculeName: Bacillus subtilis DnaB / type: sample / ID: 1000 / Oligomeric state: Homotetramer / Number unique components: 1
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa
Method: Sedimentation equilibrium by analytical ultracentrifugation

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Macromolecule #1: DnaB

MacromoleculeName: DnaB / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Bacillus subtilis (bacteria) / Cell: Bacteria / Location in cell: Plasma membrane
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pSMG37
SequenceInterPro: INTERPRO: IPR010833

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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

Concentration0.088 mg/mL
BufferpH: 7.5 / Details: 50mM Tris-HCl, 50mM NaCl, 2mM MgCl2 and 1mM DTT
StainingType: NEGATIVE
Details: Grids with adsorbed protein stained on 2% uranyl acetate for 1 minute
GridDetails: collodion/carbon coated 400 mesh copper grid
VitrificationCryogen name: NONE

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Electron microscopy

MicroscopeJEOL 2000EX
Electron beamAcceleration voltage: 80 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 60000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 21 µm / Number real images: 20 / Average electron dose: 20 e/Å2 / Bits/pixel: 8

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Xmipp and EMAN / Number images used: 8352

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