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- EMDB-2361: Electron cryo-EM of the full-length Thermus thermophilus DNA gyra... -

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Basic information

Entry
Database: EMDB / ID: EMD-2361
TitleElectron cryo-EM of the full-length Thermus thermophilus DNA gyrase in complex with a 155bp DNA and ciprofloxacin
Map dataRecontruction of the full length Thermus thermophilus DNA gyrase bound to DNA.
Sample
  • Sample: DNA-bound complex of Thermus thermophilus DNA gyrase with a 155bp DNA in presence of ADPNP and ciprofoxacin.
  • Protein or peptide: DNA gyrase
  • DNA: linear DNA
KeywordsDNA gyrase / DNA topoisomerase / DNA supercoiling
Biological speciesThermus thermophilus (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 23.0 Å
AuthorsPapillon J / Menetret JF / Batisse C / Helye R / Schultz P / Potier N / 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 13, 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: 0.55
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2361.png

Downloads & links

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Map

FileDownload / File: emd_2361.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRecontruction of the full length Thermus thermophilus DNA gyrase bound to DNA.
Voxel sizeX=Y=Z: 1.92 Å
Density
Contour LevelBy AUTHOR: 0.55 / Movie #1: 0.55
Minimum - Maximum-1.24862158 - 3.11563373
Average (Standard dev.)0.02745328 (±0.18016443)
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-1.2493.1160.027

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Supplemental data

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Sample components

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Entire : DNA-bound complex of Thermus thermophilus DNA gyrase with a 155bp...

EntireName: DNA-bound complex of Thermus thermophilus DNA gyrase with a 155bp DNA in presence of ADPNP and ciprofoxacin.
Components
  • Sample: DNA-bound complex of Thermus thermophilus DNA gyrase with a 155bp DNA in presence of ADPNP and ciprofoxacin.
  • Protein or peptide: DNA gyrase
  • DNA: linear DNA

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Supramolecule #1000: DNA-bound complex of Thermus thermophilus DNA gyrase with a 155bp...

SupramoleculeName: DNA-bound complex of Thermus thermophilus DNA gyrase with a 155bp DNA in presence of ADPNP and ciprofoxacin.
type: sample / ID: 1000 / Details: monodisperse complex / Oligomeric state: dimer / Number unique components: 1
Molecular weightExperimental: 413 KDa / Theoretical: 413 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 subinits of the DNA gyrase were fused for structural stability.
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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Macromolecule #2: linear DNA

MacromoleculeName: linear DNA / type: dna / ID: 2
Details: A 155bp DNA was used to form the DNA-bound complex. ADPNP (a non hydrolyzable analog of ATP) and ciprofloxacin (quinolone antibiotic) were added to stabilize the complex
Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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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 11, 2011
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 900 / 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: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 / Number images used: 41500

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