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

Basic information

• Entry: Database: EMDB / ID: EMD-2321
• Title: The bacterial DnaC helicase loader is a DnaB ring breaker
• Map data: Negative 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

• Keywords: Bacterial DNA replication / DNA replication initiation / helicase / helicase loader / DnaB / DnaC / electron microscopy / SAXS / clamp loader / structure

Function / homology

Function:

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 / DNA 5'-3' helicase / primosome complex / DNA replication, synthesis of primer / replisome / DNA duplex unwinding / DNA strand elongation involved in DNA replication / response to ionizing radiation / DNA unwinding involved in DNA replication / replication fork processing / DNA replication initiation / DNA helicase activity / isomerase activity / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / 5'-3' DNA helicase activity / DNA replication / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / cytosol

Domain/homology:

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

Component:

Replicative DNA helicase DnaB / Replicative helicase loader DnaC

• Biological species: Escherichia coli (E. coli)
• Method: single particle reconstruction / negative staining / Resolution: 25.0 Å
• Authors: Arias-Palomo E / O'Shea VL / Hood IV / Berger JM
• Citation: Journal: 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-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

Deposition	Feb 26, 2013	-
Header (metadata) release	Mar 13, 2013	-
Map release	Apr 17, 2013	-
Update	Apr 24, 2013	-
Current status	Apr 24, 2013	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_2321.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Negative staining reconstruction of E. coli DnaB/DnaC complex
• Voxel size: X=Y=Z: 4.36 Å

Density

Contour Level	By AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum	-5.65025902 - 13.01332378
Average (Standard dev.)	0.0 (±0.99999899)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 80 80 80 Spacing 80 80 80
Cell	A=B=C: 348.80002 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	4.36	4.36	4.36
M x/y/z	80	80	80
origin x/y/z	0.000	0.000	0.000
length x/y/z	348.800	348.800	348.800
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-36	-12	-40
NX/NY/NZ	73	25	81
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	80	80	80
D min/max/mean	-5.650	13.013	0.000

Supplemental data

Sample components

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

• Entire: Name: 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

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

• Supramolecule: Name: 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 weight: Theoretical: 480 KDa

Macromolecule #1: DnaB replicative helicase

• Macromolecule: Name: 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 weight: Theoretical: 52 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)
• Sequence: UniProtKB: Replicative DNA helicase DnaB / InterPro: DNA helicase, DnaB type

Macromolecule #2: DnaC helicase loading factor

• Macromolecule: Name: 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 weight: Theoretical: 28 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)
• Sequence: UniProtKB: Replicative helicase loader DnaC

Experimental details

Structure determination

• Method: negative staining
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 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
• Staining: Type: NEGATIVE; Details: Grids with adsorbed protein were floated on 2% w/v uranyl formate for 45 seconds
• Grid: Details: 400 mesh copper grid with thin carbon support, glow discharged for 20 seconds
• Vitrification: Cryogen name: NONE / Instrument: OTHER

Electron microscopy

• Microscope: FEI TECNAI 12
• Temperature: Average: 297 K
• Date: Jan 21, 2011
• Image recording: Category: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Average electron dose: 25 e/Ų
• Electron beam: Acceleration voltage: 120 kV / Electron source: LAB6
• Electron optics: Illumination 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 stage: Specimen holder model: SIDE ENTRY, EUCENTRIC

Image processing

• Details: The 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 correction: Details: Each micrograph
• Final reconstruction: Applied 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