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- PDB-9ggq: E.coli gyrase holocomplex with cleaved chirally wrapped 217 bp DN... -

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Basic information

Entry
Database: PDB / ID: 9ggq
TitleE.coli gyrase holocomplex with cleaved chirally wrapped 217 bp DNA fragment and moxifloxacin
Components
  • (DNA gyrase subunit ...) x 2
  • Mu217F
  • Mu217R
KeywordsISOMERASE / DNA gyrase / type II topoisomerase / moxifloxacin / DNA crossover
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 / metal ion binding / identical protein binding / membrane / cytosol / cytoplasm
Similarity search - Function
: / 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 ...: / 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 / DNA gyrase, subunit B / DNA topoisomerase, type IIA, subunit B / DNA gyrase B subunit, C-terminal / DNA gyrase B subunit, carboxyl terminus / Topoisomerase (Topo) IIA-type catalytic domain profile. / 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 / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold
Similarity search - Domain/homology
Chem-MFX / : / DNA / DNA (> 10) / DNA (> 100) / DNA gyrase subunit A / DNA gyrase subunit B
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia phage Mu (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsGhilarov, D. / Heddle, J.G. / Pabis, M.
Funding support United Kingdom, Poland, 3items
OrganizationGrant numberCountry
Wellcome Trust221868/Z/20/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/X01097X/1 United Kingdom
Polish National Science Centre2020/39/B/NZ1/02898 Poland
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Structural basis of chiral wrap and T-segment capture by DNA gyrase.
Authors: Elizabeth Michalczyk / Zuzanna Pakosz-Stępień / Jonathon D Liston / Olivia Gittins / Marta Pabis / Jonathan G Heddle / Dmitry Ghilarov /
Abstract: Type II topoisomerase DNA gyrase transduces the energy of ATP hydrolysis into the negative supercoiling of DNA. The postulated catalytic mechanism involves stabilization of a chiral DNA loop followed ...Type II topoisomerase DNA gyrase transduces the energy of ATP hydrolysis into the negative supercoiling of DNA. The postulated catalytic mechanism involves stabilization of a chiral DNA loop followed by the passage of the T-segment through the temporarily cleaved G-segment resulting in sign inversion. The molecular basis for this is poorly understood as the chiral loop has never been directly observed. We have obtained high-resolution cryoEM structures of gyrase with chirally wrapped 217 bp DNA with and without the fluoroquinolone moxifloxacin (MFX). Each structure constrains a positively supercoiled figure-of-eight DNA loop stabilized by a GyrA β-pinwheel domain which has the structure of a flat disc. By comparing the catalytic site of the native drug-free and MFX-bound gyrase structures both of which contain a single metal ion, we demonstrate that the enzyme is observed in a native precatalytic state. Our data imply that T-segment trapping is not dependent on the dimerization of the ATPase domains which appears to only be possible after strand passage has taken place.
History
DepositionAug 13, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
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Revision 1.1Nov 20, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2Dec 4, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
E: Mu217R
H: Mu217F
G: Mu217F
F: Mu217R
hetero molecules


Theoretical massNumber of molelcules
Total (without water)642,64914
Polymers641,7498
Non-polymers9006
Water1448
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA gyrase subunit ... , 2 types, 4 molecules ACBD

#1: Protein DNA gyrase subunit A


Mass: 97854.305 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: MG1655 / Gene: gyrA, hisW, nalA, parD, b2231, JW2225 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P0AES4, DNA topoisomerase (ATP-hydrolysing)
#2: Protein DNA gyrase subunit B / Type IIA topoisomerase subunit GyrB


Mass: 90891.734 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: MG1655
Gene: gyrB, acrB, cou, himB, hisU, nalC, parA, pcbA, b3699, JW5625
Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P0AES6, DNA topoisomerase (ATP-hydrolysing)

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DNA chain , 2 types, 4 molecules EFHG

#3: DNA chain Mu217R


Mass: 65926.109 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage Mu (virus) / Production host: Escherichia coli (E. coli) / References: GenBank: 215533
#4: DNA chain Mu217F


Mass: 66202.258 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage Mu (virus) / Production host: Escherichia coli (E. coli) / References: GenBank: 215533

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Non-polymers , 3 types, 14 molecules

#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-MFX / 1-cyclopropyl-6-fluoro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid / moxifloxacin


Mass: 401.431 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H24FN3O4 / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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

ComponentName: Ternary complex of Escherichia coli DNA gyrase with 217 bp linear DNA fragment and moxifloxacin
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40.68 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
8PHENIXmodel refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 79415 / Symmetry type: POINT

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