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- EMDB-2321: The bacterial DnaC helicase loader is a DnaB ring breaker -

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Basic information

Entry
Database: EMDB / ID: EMD-2321
TitleThe bacterial DnaC helicase loader is a DnaB ring breaker
Map dataNegative staining reconstruction of E. coli DnaB/DnaC complex
Sample
  • Sample: E. coli DnaB helicase bound to the DnaC loading factor
  • Protein or peptide: DnaB replicative helicase
  • Protein or peptide: DnaC helicase loading factor
KeywordsBacterial DNA replication / DNA replication initiation / helicase / helicase loader / DnaB / DnaC / electron microscopy / SAXS / clamp loader / structure
Function / homology
Function and homology information


DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / DNA 5'-3' helicase / DNA replication, synthesis of primer / replisome ...DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / DNA 5'-3' helicase / DNA replication, synthesis of primer / replisome / : / response to ionizing radiation / DNA strand elongation involved in DNA replication / replication fork processing / : / DNA replication initiation / DNA helicase activity / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / helicase activity / isomerase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / 5'-3' DNA helicase activity / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / DNA clamp loader activity / DNA replication / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / cytosol
Similarity search - Function
DNA replication protein DnaC/insertion sequence putative ATP-binding protein / IstB-like ATP-binding protein / IstB-like ATP binding protein / DNA helicase, DnaB type / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase N terminal domain / DNA helicase, DnaB-like, N-terminal domain superfamily / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / DnaB-like helicase C terminal domain ...DNA replication protein DnaC/insertion sequence putative ATP-binding protein / IstB-like ATP-binding protein / IstB-like ATP binding protein / DNA helicase, DnaB type / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase N terminal domain / DNA helicase, DnaB-like, N-terminal domain superfamily / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Replicative DNA helicase DnaB / Replicative helicase loader DnaC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsArias-Palomo E / O'Shea VL / Hood IV / Berger JM
CitationJournal: Cell / Year: 2013
Title: The bacterial DnaC helicase loader is a DnaB ring breaker.
Authors: Ernesto Arias-Palomo / Valerie L O'Shea / Iris V Hood / James M Berger /
Abstract: Dedicated AAA+ ATPases deposit hexameric ring-shaped helicases onto DNA to promote replication in cellular organisms. To understand how loading occurs, we used electron microscopy and small angle X- ...Dedicated AAA+ ATPases deposit hexameric ring-shaped helicases onto DNA to promote replication in cellular organisms. To understand how loading occurs, we used electron microscopy and small angle X-ray scattering (SAXS) to determine the ATP-bound structure of the intact E. coli DnaB⋅DnaC helicase/loader complex. The 480 kDa dodecamer forms a three-tiered assembly, in which DnaC adopts a spiral configuration that remodels N-terminal scaffolding and C-terminal motor regions of DnaB to produce a clear break in the helicase ring. Surprisingly, DnaC's AAA+ fold is dispensable for ring remodeling because the DnaC isolated helicase-binding domain can both load DnaB onto DNA and increase the efficiency by which the helicase acts on substrates in vitro. Our data demonstrate that DnaC opens DnaB by a mechanism akin to that of polymerase clamp loaders and indicate that bacterial replicative helicases, like their eukaryotic counterparts, possess autoregulatory elements that influence how hexameric motor domains are loaded onto and unwind DNA.
History
DepositionFeb 26, 2013-
Header (metadata) releaseMar 13, 2013-
Map releaseApr 17, 2013-
UpdateApr 24, 2013-
Current statusApr 24, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2321.png

Downloads & links

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Map

FileDownload / File: emd_2321.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative staining reconstruction of E. coli DnaB/DnaC complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.36 Å/pix.
x 80 pix.
= 348.8 Å		4.36 Å/pix.
x 80 pix.
= 348.8 Å		4.36 Å/pix.
x 80 pix.
= 348.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.36 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-5.65025902 - 13.01332378
Average (Standard dev.)0.0 (±0.99999899)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 348.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.364.364.36
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z348.800348.800348.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-12-40
NX/NY/NZ732581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-5.65013.0130.000

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Supplemental data

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Sample components

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Entire : E. coli DnaB helicase bound to the DnaC loading factor

EntireName: E. coli DnaB helicase bound to the DnaC loading factor
Components
  • Sample: E. coli DnaB helicase bound to the DnaC loading factor
  • Protein or peptide: DnaB replicative helicase
  • Protein or peptide: DnaC helicase loading factor

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Supramolecule #1000: E. coli DnaB helicase bound to the DnaC loading factor

SupramoleculeName: E. coli DnaB helicase bound to the DnaC loading factor
type: sample / ID: 1000
Oligomeric state: One homohexamer of DnaB binds to one homohexamer of DnaC
Number unique components: 2
Molecular weightTheoretical: 480 KDa

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Macromolecule #1: DnaB replicative helicase

MacromoleculeName: DnaB replicative helicase / type: protein_or_peptide / ID: 1 / Name.synonym: Replicative DNA helicase / Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 52 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Replicative DNA helicase DnaB / InterPro: DNA helicase, DnaB type

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Macromolecule #2: DnaC helicase loading factor

MacromoleculeName: DnaC helicase loading factor / type: protein_or_peptide / ID: 2 / Name.synonym: DNA replication protein DnaC / Number of copies: 6 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 28 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Replicative helicase loader DnaC

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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

BufferpH: 8.5
Details: 20 mM Tris-HCl pH 8.5, 200 mM NaCl, 5 % glycerol, 5 mM MgCl2, 1 mM beta-mercaptoethanol, and 1 mM ADP-BeF3
StainingType: NEGATIVE
Details: Grids with adsorbed protein were floated on 2% w/v uranyl formate for 45 seconds
GridDetails: 400 mesh copper grid with thin carbon support, glow discharged for 20 seconds
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
TemperatureAverage: 297 K
DateJan 21, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Average electron dose: 25 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 6.3 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 49000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

DetailsThe particles were selected using DoG picker as available in APPION. The contrast transfer function of the microscope for each micrograph was estimated using CTFFIND3 and phase-flipped using SPIDER. DnaBC particles were subjected to a multi-model refinement as implemented in SPARX using the 3D averages obtained from the RCT reconstructions as initial references.
CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2, SPARX / Number images used: 17942

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