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- PDB-8j8a: Cryo-EM structure of Asfv topoisomerase 2 - apo conformer IIb -

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Basic information

Entry
Database: PDB / ID: 8j8a
TitleCryo-EM structure of Asfv topoisomerase 2 - apo conformer IIb
ComponentsDNA topoisomerase 2Topoisomerase
KeywordsISOMERASE / Topoisomerase / ASFV / VIRAL PROTEIN
Function / homology
Function and homology information


DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / host cell cytoplasm / DNA binding / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
DNA topoisomerase II, eukaryotic-type / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / 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 / DNA topoisomerase, type IIA, conserved site ...DNA topoisomerase II, eukaryotic-type / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / 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 / DNA topoisomerase, type IIA, conserved site / DNA topoisomerase II signature. / TopoisomeraseII / DNA topoisomerase, type IIA, subunit B, C-terminal / DNA topoisomerase, type IIA-like domain superfamily / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S5 domain 2-type fold, subgroup
Similarity search - Domain/homology
Biological speciesAfrican swine fever virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.51 Å
AuthorsChang, C.-W. / Tsai, M.-D.
Funding support Taiwan, 1items
OrganizationGrant numberCountry
Academia Sinica (Taiwan)AS-KPQ-109-TPP2 Taiwan
CitationJournal: Commun Chem / Year: 2024
Title: A unified view on enzyme catalysis by cryo-EM study of a DNA topoisomerase.
Authors: Chiung-Wen Mary Chang / Shun-Chang Wang / Chun-Hsiung Wang / Allan H Pang / Cheng-Han Yang / Yao-Kai Chang / Wen-Jin Wu / Ming-Daw Tsai /
Abstract: The theories for substrate recognition in enzyme catalysis have evolved from lock-key to induced fit, then conformational selection, and conformational selection followed by induced fit. However, the ...The theories for substrate recognition in enzyme catalysis have evolved from lock-key to induced fit, then conformational selection, and conformational selection followed by induced fit. However, the prevalence and consensus of these theories require further examination. Here we use cryogenic electron microscopy and African swine fever virus type 2 topoisomerase (AsfvTop2) to demonstrate substrate binding theories in a joint and ordered manner: catalytic selection by the enzyme, conformational selection by the substrates, then induced fit. The apo-AsfvTop2 pre-exists in six conformers that comply with the two-gate mechanism directing DNA passage and release in the Top2 catalytic cycle. The structures of AsfvTop2-DNA-inhibitor complexes show that substantial induced-fit changes occur locally from the closed apo-conformer that however is too far-fetched for the open apo-conformer. Furthermore, the ATPase domain of AsfvTop2 in the MgAMP-PNP-bound crystal structures coexist in reduced and oxidized forms involving a disulfide bond, which can regulate the AsfvTop2 function.
History
DepositionMay 1, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA topoisomerase 2
B: DNA topoisomerase 2


Theoretical massNumber of molelcules
Total (without water)272,8452
Polymers272,8452
Non-polymers00
Water0
1


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

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Components

#1: Protein DNA topoisomerase 2 / Topoisomerase


Mass: 136422.578 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) African swine fever virus / Gene: P1192R / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A0A1E3Q0

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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: ASFV Topoisomerase 2 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: African swine fever virus
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
SpecimenConc.: 0.3 mg/ml / Embedding 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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8900

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 393995 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingDetails: AsfvTop2 conformer IIa / Source name: Other / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312516
ELECTRON MICROSCOPYf_angle_d0.47516911
ELECTRON MICROSCOPYf_dihedral_angle_d4.0141671
ELECTRON MICROSCOPYf_chiral_restr0.041827
ELECTRON MICROSCOPYf_plane_restr0.0042157

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