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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 / homologyDNA gyrase, subunit A / DNA gyrase/topoisomerase IV, subunit A, C-terminal repeat / DNA gyrase C-terminal domain, beta-propeller / DNA gyrase/topoisomerase IV, subunit A / DNA gyrase/topoisomerase IV, subunit A, C-terminal / DNA topoisomerase, type IIA-like domain superfamily / DNA topoisomerase, type IIA, subunit A/ C-terminal, alpha-beta / DNA topoisomerase, type IIA, subunit A/C-terminal / Type IIA DNA topoisomerase subunit A, alpha-helical domain superfamily / DNA topoisomerase type II (ATP-hydrolyzing) activity ...DNA gyrase, subunit A / DNA gyrase/topoisomerase IV, subunit A, C-terminal repeat / DNA gyrase C-terminal domain, beta-propeller / DNA gyrase/topoisomerase IV, subunit A / DNA gyrase/topoisomerase IV, subunit A, C-terminal / DNA topoisomerase, type IIA-like domain superfamily / DNA topoisomerase, type IIA, subunit A/ C-terminal, alpha-beta / DNA topoisomerase, type IIA, subunit A/C-terminal / Type IIA DNA topoisomerase subunit A, alpha-helical domain superfamily / DNA topoisomerase type II (ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / DNA-dependent DNA replication / chromosome / DNA binding / ATP binding / cytoplasm / DNA gyrase subunit A
Function and homology information
Specimen sourceStreptococcus pneumoniae G54 (bacteria)
Cloning vector pBR322 (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 6.35 Å resolution
AuthorsSoczek, K.M. / Grant, T. / Rosenthal, P.B. / Mondragon, A.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 10, 2018 / Release: Dec 5, 2018

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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
Polyers490,92110
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide
DNA gyrase subunit A /


Mass: 57977.578 Da / Num. of mol.: 8 / Fragment: UNP residues 20-506
Source: (gene. exp.) Streptococcus pneumoniae G54 (bacteria)
Gene: gyrA_1, gyrA, ERS409327_01712 / Plasmid name: pMCSG7 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A0Y2BJX7, DNA topoisomerase (ATP-hydrolysing)
#2: DNA chain DNA (44-MER)


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


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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 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 symmetryCOMPLEX1, 2, 30MULTIPLE SOURCES
2DNA Gyrase ACOMPLEX11RECOMBINANT
3DNACOMPLEX2, 31NATURAL
Molecular weightValue: 0.49 MDa / Experimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
12512566Streptococcus pneumoniae G54 (bacteria)
2347470Cloning vector pBR322 (others)
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 / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277

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Electron microscopy imaging

MicroscopyMicroscope model: JEOL 3200FS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 30000 / Calibrated magnification: 40323 / Nominal defocus max: 3000 / Nominal defocus min: 1000 / Cs: 2 / C2 aperture diameter: 5 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Temperature (max): 100 / Temperature (min): 100
Image recordingAverage exposure time: 12 / Electron dose: 62 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of real images: 718
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20
Image scansSampling size: 5 / 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 selectionNumber of particles selected: 387297
SymmetryPoint symmetry: C2
3D reconstructionResolution: 6.35 / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 30637 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 4Z2C
Pdb chain ID: A / Pdb chain residue range: 2-486

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