+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2322 | |||||||||
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Title | The bacterial DnaC helicase loader is a DnaB ring breaker | |||||||||
Map data | negative staining reconstruction of E. coli DnaB | |||||||||
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 / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / DNA 5'-3' helicase / primosome complex / 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 / DNA 5'-3' helicase / primosome complex / DNA replication, synthesis of primer / replisome / DNA duplex unwinding / response to ionizing radiation / DNA unwinding involved in DNA replication / replication fork processing / DNA replication initiation / DNA helicase activity / helicase activity / 5'-3' DNA helicase activity / 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_2322.map.gz | 1.7 MB | EMDB map data format | |
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Header (meta data) | emd-2322-v30.xml emd-2322.xml | 8.9 KB 8.9 KB | Display Display | EMDB header |
Images | emd_2322.png | 73.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2322 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2322 | HTTPS FTP |
-Validation report
Summary document | emd_2322_validation.pdf.gz | 203.4 KB | Display | EMDB validaton report |
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Full document | emd_2322_full_validation.pdf.gz | 202.5 KB | Display | |
Data in XML | emd_2322_validation.xml.gz | 5.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2322 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2322 | 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_2322.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 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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 replicative helicase
Entire | Name: E. coli DnaB replicative helicase |
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Components |
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-Supramolecule #1000: E. coli DnaB replicative helicase
Supramolecule | Name: E. coli DnaB replicative helicase / type: sample / ID: 1000 / Oligomeric state: Homohexamer / Number unique components: 1 |
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Molecular weight | Theoretical: 312 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 DnaB |
-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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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 |
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. |
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CTF correction | Details: Each micrograph |
Final reconstruction | Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2, SPARX / Number images used: 9617 |