+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2321 | |||||||||
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Title | The bacterial DnaC helicase loader is a DnaB ring breaker | |||||||||
Map data | Negative staining reconstruction of E. coli DnaB/DnaC complex | |||||||||
Sample |
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Keywords | Bacterial 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 / DNA helicase complex / DnaB-DnaC-DnaT-PriA-PriB complex / primosome complex / replisome / DNA replication, synthesis of primer / DNA strand elongation involved in DNA replication ...DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DNA helicase complex / DnaB-DnaC-DnaT-PriA-PriB complex / primosome complex / replisome / DNA replication, synthesis of primer / DNA strand elongation involved in DNA replication / DNA duplex unwinding / response to ionizing radiation / replication fork processing / DNA unwinding involved in DNA replication / DNA replication initiation / DNA helicase activity / helicase activity / DNA helicase / DNA replication / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / cytosol Similarity search - Function | |||||||||
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- ...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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2321.map.gz | 1.7 MB | EMDB map data format | |
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Header (meta data) | emd-2321-v30.xml emd-2321.xml | 9.9 KB 9.9 KB | Display Display | EMDB header |
Images | emd_2321.png | 76.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2321 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2321 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2321.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Negative staining reconstruction of E. coli DnaB/DnaC complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.36 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-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 |
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Components |
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-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 |
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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 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 52 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | UniProtKB: Replicative DNA helicase / 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 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 28 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | UniProtKB: DNA replication protein DnaC |
-Experimental details
-Structure determination
Method | negative staining |
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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 |
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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 |
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Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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 |
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/Å2 |
-Image processing
CTF correction | Details: Each micrograph |
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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 |
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. |