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- PDB-6zy5: Cryo-EM structure of the Human topoisomerase II alpha DNA-binding... -

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Basic information

Entry
Database: PDB / ID: 6zy5
TitleCryo-EM structure of the Human topoisomerase II alpha DNA-binding/cleavage domain in State 1
Components
  • DNA (5'-D(*CP*GP*CP*GP*CP*AP*TP*CP*GP*TP*CP*AP*TP*CP*CP*TP*C)-3')
  • DNA (5'-D(*GP*AP*GP*GP*AP*TP*GP*AP*CP*GP*AP*TP*G)-3')
  • DNA topoisomerase 2-alpha
KeywordsISOMERASE / Human Topoisomerase / Etoposide / DNA
Function / homology
Function and homology information


: / positive regulation of single stranded viral RNA replication via double stranded DNA intermediate / apoptotic chromosome condensation / sister chromatid segregation / resolution of meiotic recombination intermediates / female meiotic nuclear division / embryonic cleavage / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex / : / Transcription of E2F targets under negative control by DREAM complex ...: / positive regulation of single stranded viral RNA replication via double stranded DNA intermediate / apoptotic chromosome condensation / sister chromatid segregation / resolution of meiotic recombination intermediates / female meiotic nuclear division / embryonic cleavage / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex / : / Transcription of E2F targets under negative control by DREAM complex / DNA binding, bending / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / SUMOylation of DNA replication proteins / chromosome, centromeric region / hematopoietic progenitor cell differentiation / ATP-dependent activity, acting on DNA / condensed chromosome / ubiquitin binding / male germ cell nucleus / chromosome segregation / protein kinase C binding / regulation of circadian rhythm / rhythmic process / positive regulation of apoptotic process / ribonucleoprotein complex / protein heterodimerization activity / DNA damage response / chromatin binding / nucleolus / magnesium ion binding / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / protein-containing complex / DNA binding / RNA binding / nucleoplasm / ATP binding / nucleus / cytoplasm
Similarity search - Function
DTHCT / DTHCT (NUC029) region / DNA topoisomerase 2, TOPRIM domain / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / DNA topoisomerase II, eukaryotic-type / : / Topoisomerase (Topo) IIA-type catalytic domain profile. / DNA topoisomerase, type IIA, alpha-helical domain superfamily / DNA topoisomerase, type IIA, domain A ...DTHCT / DTHCT (NUC029) region / DNA topoisomerase 2, TOPRIM domain / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / DNA topoisomerase II, eukaryotic-type / : / 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/HSP90-like ATPase / 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-EVP / DNA / DNA (> 10) / DNA topoisomerase 2-alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 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 Biology (FRISBI)FRISBI ANR-10-INBS-05 France
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis for allosteric regulation of Human Topoisomerase IIα.
Authors: Arnaud Vanden Broeck / Christophe Lotz / Robert Drillien / Léa Haas / Claire Bedez / Valérie Lamour /
Abstract: The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in ...The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6-7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme's activities and opens perspective for the analysis of post-translational modifications.
History
DepositionJul 30, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 26, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 2, 2021Group: 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 / _citation.title / _citation_author.identifier_ORCID
Revision 1.2May 1, 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
B: DNA topoisomerase 2-alpha
D: DNA (5'-D(*CP*GP*CP*GP*CP*AP*TP*CP*GP*TP*CP*AP*TP*CP*CP*TP*C)-3')
E: DNA (5'-D(*GP*AP*GP*GP*AP*TP*GP*AP*CP*GP*AP*TP*G)-3')
F: DNA (5'-D(*CP*GP*CP*GP*CP*AP*TP*CP*GP*TP*CP*AP*TP*CP*CP*TP*C)-3')
C: DNA (5'-D(*GP*AP*GP*GP*AP*TP*GP*AP*CP*GP*AP*TP*G)-3')
A: DNA topoisomerase 2-alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)368,8468
Polymers367,6696
Non-polymers1,1772
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18290 Å2
ΔGint-83 kcal/mol
Surface area84250 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_1ens_2chain "C"
d_2ens_2chain "E"
d_1ens_3chain "D"
d_2ens_3chain "F"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ASNARGH1 - 760
d_21ens_1ASNARGA1 - 760
d_11ens_2DGDGG
d_21ens_2DGDGE
d_11ens_3DCDCB
d_21ens_3DCDCF

NCS ensembles :
ID
ens_1
ens_2
ens_3

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Components

#1: Protein DNA topoisomerase 2-alpha / DNA topoisomerase II / alpha isozyme


Mass: 174654.406 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TOP2A, TOP2 / Cell line (production host): BHK21 / Production host: Vaccinia virus Ankara
References: UniProt: P11388, DNA topoisomerase (ATP-hydrolysing)
#2: DNA chain DNA (5'-D(*CP*GP*CP*GP*CP*AP*TP*CP*GP*TP*CP*AP*TP*CP*CP*TP*C)-3')


Mass: 5099.298 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: DNA chain DNA (5'-D(*GP*AP*GP*GP*AP*TP*GP*AP*CP*GP*AP*TP*G)-3')


Mass: 4080.671 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: Chemical ChemComp-EVP / (5S,5aR,8aR,9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol -5-yl 4,6-O-[(1R)-ethylidene]-beta-D-glucopyranoside / Etoposide / VP-16


Mass: 588.557 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C29H32O13 / Comment: medication, chemotherapy*YM
Has ligand of interestN

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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
1Etoposide-bound Human Top2a DNA-binding/cleavage domain in state 1COMPLEX#1-#30RECOMBINANT
2DNA topoisomerase 2-alphaCOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.205 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
22Vaccinia virus Ankara126794BHK21
33synthetic construct (others)32630
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 0.01 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.18rc4_3812: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4GctfCTF correction
5RELION3.1CTF correction
6cryoSPARCv3CTF correction
9UCSF Chimeramodel fitting
11cryoSPARCv3initial Euler assignment
12cryoSPARCv3final Euler assignment
14cryoSPARCv33D reconstruction
15PHENIX1.18rc4model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1908092
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60601 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5GWK

5gwk


Accession code: 5GWK / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 94.08 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01113908
ELECTRON MICROSCOPYf_angle_d1.15719030
ELECTRON MICROSCOPYf_dihedral_angle_d14.5965426
ELECTRON MICROSCOPYf_chiral_restr0.0852094
ELECTRON MICROSCOPYf_plane_restr0.0072220
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2HFELECTRON MICROSCOPYNCS constraints0.0358826580513
ens_2d_2GDELECTRON MICROSCOPYNCS constraints0.0444486391381
ens_3d_2ABELECTRON MICROSCOPYNCS constraints0.0590167148566

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