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- EMDB-2360: Electron cryo-EM of full-length Thermus thermophilus DNA gyrase -

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Basic information

Entry
Database: EMDB / ID: EMD-2360
TitleElectron cryo-EM of full-length Thermus thermophilus DNA gyrase
Map dataReconstruction of full length Thermus thermophilus DNA gyrase with ADPNP (non hydrolyzable analog of ATP)
Sample
  • Sample: Holoenzyme complex of Thermus thermophilus DNA gyrase with ADPNP
  • Protein or peptide: DNA gyrase
KeywordsDNA topoisomerase / DNA gyrase
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.8 Å
AuthorsPapillon J / Menetret JF / Batisse C / Helye R / Schultz P / Potier P / Lamour V
CitationJournal: Nucleic Acids Res / Year: 2013
Title: Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase.
Authors: Julie Papillon / Jean-François Ménétret / Claire Batisse / Reynald Hélye / Patrick Schultz / Noëlle Potier / Valérie Lamour /
Abstract: Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA ...Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA through a transient break formed in another duplex. The bacterial DNA gyrase, a target for broad-spectrum antibiotics, is the sole Topo2A enzyme able to introduce negative supercoils. We reveal here for the first time the architecture of the full-length Thermus thermophilus DNA gyrase alone and in a cleavage complex with a 155 bp DNA duplex in the presence of the antibiotic ciprofloxacin, using cryo-electron microscopy. The structural organization of the subunits of the full-length DNA gyrase points to a central role of the ATPase domain acting like a 'crossover trap' that may help to sequester the DNA positive crossover before strand passage. Our structural data unveil how DNA is asymmetrically wrapped around the gyrase-specific C-terminal β-pinwheel domains and guided to introduce negative supercoils through cooperativity between the ATPase and β-pinwheel domains. The overall conformation of the drug-induced DNA binding-cleavage complex also suggests that ciprofloxacin traps a DNA pre-transport conformation.
History
DepositionApr 12, 2013-
Header (metadata) releaseMay 22, 2013-
Map releaseJul 10, 2013-
UpdateSep 18, 2013-
Current statusSep 18, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_2360.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of full length Thermus thermophilus DNA gyrase with ADPNP (non hydrolyzable analog of ATP)
Voxel sizeX=Y=Z: 1.92 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.61629558 - 2.332021
Average (Standard dev.)0.0179102 (±0.14213429)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 368.63998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.921.921.92
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z368.640368.640368.640
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.6162.3320.018

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Supplemental data

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Sample components

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Entire : Holoenzyme complex of Thermus thermophilus DNA gyrase with ADPNP

EntireName: Holoenzyme complex of Thermus thermophilus DNA gyrase with ADPNP
Components
  • Sample: Holoenzyme complex of Thermus thermophilus DNA gyrase with ADPNP
  • Protein or peptide: DNA gyrase

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Supramolecule #1000: Holoenzyme complex of Thermus thermophilus DNA gyrase with ADPNP

SupramoleculeName: Holoenzyme complex of Thermus thermophilus DNA gyrase with ADPNP
type: sample / ID: 1000 / Details: monodisperse complex formed in presence of ADPNP / Oligomeric state: dimer / Number unique components: 1
Molecular weightExperimental: 321 KDa / Theoretical: 321 KDa / Method: native mass spectrometry

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Macromolecule #1: DNA gyrase

MacromoleculeName: DNA gyrase / type: protein_or_peptide / ID: 1 / Name.synonym: bacterial DNA topoisomerase 2A
Details: The two subunits of the DNA gyrase were fused for structural stability. ADPNP (non hydrolysable analog of ATP) was added to form the holoenzyme complex. This complex was crosslinked with ...Details: The two subunits of the DNA gyrase were fused for structural stability. ADPNP (non hydrolysable analog of ATP) was added to form the holoenzyme complex. This complex was crosslinked with glutaraldehyde prior to vitrification.
Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightExperimental: 321 KDa / Theoretical: 321 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: modified pET28a

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.150 mg/mL
BufferpH: 8 / Details: 20mM Hepes , 100 mM NaCl, 5mM MgCl2, 1mM DTT
GridDetails: Quantifoil R 2/2 holey carbon copper grids
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Method: Plunging immediately after blotting

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 59000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: -1.0 µm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateDec 10, 2011
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 600 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: phase flipping (each particle)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 / Number images used: 20500

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