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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2360 | |||||||||
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Title | Electron cryo-EM of full-length Thermus thermophilus DNA gyrase | |||||||||
![]() | Reconstruction of full length Thermus thermophilus DNA gyrase with ADPNP (non hydrolyzable analog of ATP) | |||||||||
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![]() | DNA topoisomerase / DNA gyrase | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 16.8 Å | |||||||||
![]() | Papillon J / Menetret JF / Batisse C / Helye R / Schultz P / Potier P / Lamour V | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 2.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 9.2 KB 9.2 KB | Display Display | ![]() |
Images | ![]() | 52.1 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 184.3 KB | Display | ![]() |
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Full document | ![]() | 183.4 KB | Display | |
Data in XML | ![]() | 6 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | Reconstruction of full length Thermus thermophilus DNA gyrase with ADPNP (non hydrolyzable analog of ATP) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.92 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Holoenzyme complex of Thermus thermophilus DNA gyrase with ADPNP
Entire | Name: Holoenzyme complex of Thermus thermophilus DNA gyrase with ADPNP |
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Components |
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-Supramolecule #1000: Holoenzyme complex of Thermus thermophilus DNA gyrase with ADPNP
Supramolecule | Name: 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 |
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Molecular weight | Experimental: 321 KDa / Theoretical: 321 KDa / Method: native mass spectrometry |
-Macromolecule #1: DNA gyrase
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 321 KDa / Theoretical: 321 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.150 mg/mL |
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Buffer | pH: 8 / Details: 20mM Hepes , 100 mM NaCl, 5mM MgCl2, 1mM DTT |
Grid | Details: Quantifoil R 2/2 holey carbon copper grids |
Vitrification | Cryogen 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
Microscope | FEI TECNAI F30 |
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Date | Dec 10, 2011 |
Image recording | Category: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 600 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 100 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 59000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: -1.0 µm |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | ![]() Model: Tecnai F30 / Image courtesy: FEI Company |
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Image processing
CTF correction | Details: phase flipping (each particle) |
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Final reconstruction | Applied 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 |