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- PDB-6rku: E. coli DNA Gyrase - DNA binding and cleavage domain in State 1 -

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Basic information

Entry
Database: PDB / ID: 6rku
TitleE. coli DNA Gyrase - DNA binding and cleavage domain in State 1
Components
  • DNA Strand 1
  • DNA Strand 2
  • DNA gyrase subunit A
  • DNA gyrase subunit B
KeywordsISOMERASE / Complex / DNA Gyrase / Inhibitor / DNA BINDING PROTEIN
Function / homology
Function and homology information


negative regulation of DNA-templated DNA replication / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex / DNA negative supercoiling activity / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / ATP-dependent activity, acting on DNA / DNA-templated DNA replication / chromosome / response to xenobiotic stimulus ...negative regulation of DNA-templated DNA replication / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex / DNA negative supercoiling activity / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / ATP-dependent activity, acting on DNA / DNA-templated DNA replication / chromosome / response to xenobiotic stimulus / response to antibiotic / DNA-templated transcription / DNA binding / ATP binding / identical protein binding / membrane / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Ubiquitin-like (UB roll) - #690 / GMP Synthetase; Chain A, domain 3 - #370 / : / GyrB, hook / DNA gyrase subunit B insert domain / DNA gyrase B subunit insert domain / DNA gyrase, subunit A / DNA gyrase/topoisomerase IV, subunit A, C-terminal repeat / DNA gyrase/topoisomerase IV, subunit A, C-terminal / : ...Ubiquitin-like (UB roll) - #690 / GMP Synthetase; Chain A, domain 3 - #370 / : / GyrB, hook / DNA gyrase subunit B insert domain / DNA gyrase B subunit insert domain / DNA gyrase, subunit A / DNA gyrase/topoisomerase IV, subunit A, C-terminal repeat / DNA gyrase/topoisomerase IV, subunit A, C-terminal / : / DNA gyrase C-terminal domain, beta-propeller / DNA gyrase subunit B, TOPRIM domain / Topoisomerase (Topo) IIA-type catalytic domain profile. / DNA gyrase, subunit B / DNA topoisomerase, type IIA, subunit B / DNA gyrase B subunit, C-terminal / DNA gyrase B subunit, carboxyl terminus / DNA topoisomerase, type IIA, alpha-helical domain superfamily / DNA topoisomerase, type IIA, domain A / DNA topoisomerase, type IIA, domain A, alpha-beta / DNA gyrase/topoisomerase IV, subunit A / DNA Topoisomerase IV / DNA topoisomerase, type IIA, subunit B, domain 2 / DNA gyrase B / DNA topoisomerase, type IIA / DNA topoisomerase, type IIA, conserved site / DNA topoisomerase II signature. / TopoisomeraseII / DNA topoisomerase, type IIA, subunit B, C-terminal / Toprim domain / DNA topoisomerase, type IIA-like domain superfamily / Toprim domain profile. / TOPRIM domain / GMP Synthetase; Chain A, domain 3 / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / Ubiquitin-like (UB roll) / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / Roll / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-JHN / DNA / DNA (> 10) / DNA gyrase subunit A / DNA gyrase subunit B
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsVanden Broeck, A. / Lamour, V.
Funding support France, 3items
OrganizationGrant numberCountry
French National Research AgencyANR-10-LABX-0030-INRT France
French National Research AgencyANR-10-IDEX-0002-02 France
French Infrastructure for Integrated Structural BiologyFRISBI ANR-10-INBS-05 France
CitationJournal: Nat Commun / Year: 2019
Title: Cryo-EM structure of the complete E. coli DNA gyrase nucleoprotein complex.
Authors: Arnaud Vanden Broeck / Christophe Lotz / Julio Ortiz / Valérie Lamour /
Abstract: DNA gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, a detailed architecture of ...DNA gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, a detailed architecture of the full-length DNA gyrase from the model organism E. coli is still missing. Herein, we report the complete structure of the E. coli DNA gyrase nucleoprotein complex trapped by the antibiotic gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of two states of the DNA-binding/cleavage domain provides a better understanding of the allosteric movements of the enzyme complex. The local atomic resolution in the DNA-bound area reaching up to 3.0 Å enables the identification of the antibiotic density. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates.
History
DepositionApr 30, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 6, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed
Revision 1.2Nov 18, 2020Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.phase_plate
Revision 1.3May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-4910
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: DNA gyrase subunit A
B: DNA gyrase subunit B
C: DNA gyrase subunit A
D: DNA gyrase subunit B
F: DNA Strand 1
E: DNA Strand 1
G: DNA Strand 2
H: DNA Strand 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)394,3649
Polymers393,9168
Non-polymers4491
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, GyrA2B2
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area22000 Å2
ΔGint-82 kcal/mol
Surface area103300 Å2
MethodPISA

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Components

#1: Protein DNA gyrase subunit A


Mass: 97088.586 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: GyrA subunit DNA Binding and cleavage domain
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: gyrA, hisW, nalA, parD, b2231, JW2225 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0AES4, DNA 5'-3' helicase
#2: Protein DNA gyrase subunit B / Type IIA topoisomerase subunit GyrB


Mass: 90073.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: GyrB subunit DNA Binding and cleavage domain
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: gyrB, acrB, cou, himB, hisU, nalC, parA, pcbA, b3699, JW5625
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0AES6, DNA 5'-3' helicase
#3: DNA chain DNA Strand 1


Mass: 4288.818 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: DNA chain DNA Strand 2


Mass: 5506.578 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#5: Chemical ChemComp-JHN / (3~{R})-3-[[4-(3,4-dihydro-2~{H}-pyrano[2,3-c]pyridin-6-ylmethylamino)piperidin-1-yl]methyl]-1,4,7-triazatricyclo[6.3.1.0^{4,12}]dodeca-6,8(12),9-triene-5,11-dione


Mass: 448.518 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H28N6O3 / Comment: antibiotic, inhibitor*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1DNA binding and cleavage domain of the E. coli DNA Gyrase in State 1COMPLEX#1-#40RECOMBINANT
2DNA GyraseCOMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#41RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli K-12 (bacteria)83333
33Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33synthetic construct (others)32630
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 800 nm / Nominal defocus min: 300 nm / Cs: 0.01 mm
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV / Phase plate: VOLTA PHASE PLATE
Image scansMovie frames/image: 40 / Used frames/image: 3-40

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4RELION2.0.3CTF correction
7UCSF Chimera1.11.2model fitting
9PHENIX1.12model refinement
10cryoSPARCV1initial Euler assignment
11RELION2.0.3final Euler assignment
13RELION2.0.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1572962
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60548 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model building

3D fitting-ID: 1 / Accession code: 3NUH / Initial refinement model-ID: 1 / PDB-ID: 3NUH

3nuh

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
3C
4D
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315924
ELECTRON MICROSCOPYf_angle_d0.53521802
ELECTRON MICROSCOPYf_dihedral_angle_d12.9349552
ELECTRON MICROSCOPYf_chiral_restr0.0392450
ELECTRON MICROSCOPYf_plane_restr0.0212694

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