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- PDB-6n1p: Dihedral oligomeric complex of GyrA N-terminal fragment with DNA,... -

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Basic information

Entry
Database: PDB / ID: 6n1p
TitleDihedral oligomeric complex of GyrA N-terminal fragment with DNA, solved by cryoEM in C2 symmetry
Components
  • (DNA (44-MER)) x 2
  • DNA gyrase subunit A
KeywordsISOMERASE/DNA / topoisomerase / oligomeric complex / DNA complex / gyrase / T-segment / ISOMERASE-DNA complex
Function / homology
Function and homology information


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 / DNA-templated DNA replication / chromosome / DNA binding / ATP binding / cytoplasm
Similarity search - Function
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 / 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 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 / 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-like domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA gyrase subunit A / DNA gyrase subunit A
Similarity search - Component
Biological speciesStreptococcus pneumoniae G54 (bacteria)
Cloning vector pBR322 (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.35 Å
AuthorsSoczek, K.M. / Grant, T. / Rosenthal, P.B. / Mondragon, A.
Funding support United States, United Kingdom, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM051350 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM118108 United States
Cancer Research UKFC001143 United Kingdom
Medical Research Council (MRC, United Kingdom)FC001143 United Kingdom
Wellcome TrustFC001143 United Kingdom
CitationJournal: Elife / Year: 2018
Title: CryoEM structures of open dimers of gyrase A in complex with DNA illuminate mechanism of strand passage.
Authors: Katarzyna M Soczek / Tim Grant / Peter B Rosenthal / Alfonso Mondragón /
Abstract: Gyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of ...Gyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events. Structures where the transported DNA molecule (T-segment) is trapped by the A subunit have not been observed. Here we present the cryoEM structures of two oligomeric complexes of open gyrase A dimers and DNA. The protein subunits in these complexes were solved to 4 Å and 5.2 Å resolution. One of the complexes traps a linear DNA molecule, a putative T-segment, which interacts with the open gyrase A dimers in two states, representing steps either prior to or after passage through the DNA-gate. The structures locate the T-segment in important intermediate conformations of the catalytic cycle and provide insights into gyrase-DNA interactions and mechanism.
History
DepositionNov 10, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 5, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Mar 20, 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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Assembly

Deposited unit
A: DNA gyrase subunit A
B: DNA gyrase subunit A
C: DNA gyrase subunit A
D: DNA gyrase subunit A
E: DNA gyrase subunit A
F: DNA gyrase subunit A
G: DNA gyrase subunit A
H: DNA gyrase subunit A
I: DNA (44-MER)
J: DNA (44-MER)


Theoretical massNumber of molelcules
Total (without water)490,92110
Polymers490,92110
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
DNA gyrase subunit A


Mass: 57977.578 Da / Num. of mol.: 8 / Fragment: UNP residues 20-506
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae G54 (bacteria)
Gene: gyrA_1, gyrA, ERS409327_01712 / Plasmid: pMCSG7 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A0Y2BJX7, UniProt: Q8DPM2*PLUS, EC: 5.99.1.3
#2: DNA chain DNA (44-MER)


Mass: 13641.771 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Cloning vector pBR322 (others)
#3: DNA chain DNA (44-MER)


Mass: 13458.600 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Cloning vector pBR322 (others)

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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
1Complex of DNA Gyrase A subunit with DNA in C2 symmetryCOMPLEXall0MULTIPLE SOURCES
2DNA Gyrase ACOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31NATURAL
Molecular weightValue: 0.49 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Streptococcus pneumoniae G54 (bacteria)512566
23Cloning vector pBR322 (others)47470
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pMCSG7
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris1
255 mMpotassium chlorideKCl1
310 mMstrontium chlorideSrCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: JEOL 3200FS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 30000 X / Calibrated magnification: 40323 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Temperature (max): 100 K / Temperature (min): 100 K
Image recordingAverage exposure time: 12 sec. / Electron dose: 62 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 718
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
2Leginon3.2image acquisition
4CTFFIND4.1CTF correction
7MDFF0.4model fitting
9RELION1.4initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13REFMAC5model refinement
14PHENIX1.12-2829model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 387297
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 6.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30637 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 4Z2C

4z2c


Pdb chain-ID: A / Accession code: 4Z2C / Pdb chain residue range: 2-486 / Source name: PDB / Type: experimental model

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