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- EMDB-2508: Nucleotide and partner-protein control of bacterial replicative h... -

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Basic information

Entry
Database: EMDB / ID: EMD-2508
TitleNucleotide and partner-protein control of bacterial replicative helicase structure and function
Map dataAquifex aeolicus DnaB replicative helicase
Sample
  • Sample: Aquifex aeolicus DnaB replicative helicase
  • Protein or peptide: Replicative DNA helicase
KeywordsDnaB / helicase / RecA / DNA replication / ATPase / Aquifex aeolicus
Function / homology
Function and homology information


primosome complex / DNA replication, synthesis of RNA primer / DNA duplex unwinding / DNA unwinding involved in DNA replication / DNA helicase activity / DNA helicase / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding / cytosol
Similarity search - Function
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
Similarity search - Component
Biological speciesAquifex aeolicus (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsStrycharska MS / Arias-Palomo E / Lyubimov AY / Erzberger JP / O'Shea VL / Bustamante C / Berger JM
CitationJournal: Mol Cell / Year: 2013
Title: Nucleotide and partner-protein control of bacterial replicative helicase structure and function.
Authors: Melania S Strycharska / Ernesto Arias-Palomo / Artem Y Lyubimov / Jan P Erzberger / Valerie L O'Shea / Carlos J Bustamante / James M Berger /
Abstract: Cellular replication forks are powered by ring-shaped, hexameric helicases that encircle and unwind DNA. To better understand the molecular mechanisms and control of these enzymes, we used multiple ...Cellular replication forks are powered by ring-shaped, hexameric helicases that encircle and unwind DNA. To better understand the molecular mechanisms and control of these enzymes, we used multiple methods to investigate the bacterial replicative helicase, DnaB. A 3.3 Å crystal structure of Aquifex aeolicus DnaB, complexed with nucleotide, reveals a newly discovered conformational state for this motor protein. Electron microscopy and small angle X-ray scattering studies confirm the state seen crystallographically, showing that the DnaB ATPase domains and an associated N-terminal collar transition between two physical states in a nucleotide-dependent manner. Mutant helicases locked in either collar state are active but display different capacities to support critical activities such as duplex translocation and primase-dependent RNA synthesis. Our findings establish the DnaB collar as an autoregulatory hub that controls the ability of the helicase to transition between different functional states in response to both nucleotide and replication initiation/elongation factors.
History
DepositionNov 14, 2013-
Header (metadata) releaseNov 20, 2013-
Map releaseJan 8, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.35
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.35
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2508.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAquifex aeolicus DnaB replicative helicase
Voxel sizeX=Y=Z: 4.36 Å
Density
Contour LevelBy AUTHOR: 2.35 / Movie #1: 2.35
Minimum - Maximum-3.08688807 - 10.69586468
Average (Standard dev.)0.17210713 (±0.90351921)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 279.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.364.364.36
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z279.040279.040279.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-3.08710.6960.172

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Supplemental data

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Sample components

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Entire : Aquifex aeolicus DnaB replicative helicase

EntireName: Aquifex aeolicus DnaB replicative helicase
Components
  • Sample: Aquifex aeolicus DnaB replicative helicase
  • Protein or peptide: Replicative DNA helicase

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Supramolecule #1000: Aquifex aeolicus DnaB replicative helicase

SupramoleculeName: Aquifex aeolicus DnaB replicative helicase / type: sample / ID: 1000 / Oligomeric state: hexamer / Number unique components: 1
Molecular weightTheoretical: 322 KDa

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Macromolecule #1: Replicative DNA helicase

MacromoleculeName: Replicative DNA helicase / type: protein_or_peptide / ID: 1 / Name.synonym: DnaB / Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Aquifex aeolicus (bacteria)
Molecular weightTheoretical: 53.6 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Replicative DNA helicase

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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: 7.5
Details: 50 mM HEPES pH 7.5, 200 mM KCl, 5% Glycerol, 5 mM MgCl2, 1 mM ADP-BeF3
StainingType: NEGATIVE / Details: 2% (w/v) uranyl acetate
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination 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 stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateJun 29, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 25 e/Å2

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Number images used: 38702

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