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- EMDB-11552: Entire Human Top2a - Interface between ATPase and DNA-binding/cle... -

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Basic information

Entry
Database: EMDB / ID: EMD-11552
TitleEntire Human Top2a - Interface between ATPase and DNA-binding/cleavage domains
Map data
Sample
  • Complex: Etoposide-bound Entire Human Top2a
    • Protein or peptide: Human Top2a
Function / homology
Function and homology information


negative regulation of DNA duplex unwinding / positive regulation of single stranded viral RNA replication via double stranded DNA intermediate / sister chromatid segregation / apoptotic chromosome condensation / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex / resolution of meiotic recombination intermediates / female meiotic nuclear division / DNA ligation / embryonic cleavage / Transcription of E2F targets under negative control by DREAM complex ...negative regulation of DNA duplex unwinding / positive regulation of single stranded viral RNA replication via double stranded DNA intermediate / sister chromatid segregation / apoptotic chromosome condensation / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex / resolution of meiotic recombination intermediates / female meiotic nuclear division / DNA ligation / embryonic cleavage / Transcription of E2F targets under negative control by DREAM complex / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA binding, bending / DNA topological change / SUMOylation of DNA replication proteins / chromosome, centromeric region / ATP-dependent activity, acting on DNA / hematopoietic progenitor cell differentiation / 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 / DNA topoisomerase II, eukaryotic-type / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / 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 ...DTHCT / DTHCT (NUC029) region / DNA topoisomerase 2, TOPRIM domain / DNA topoisomerase II, eukaryotic-type / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / 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 / DNA topoisomerase, type IIA-like domain superfamily / Toprim domain / 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
DNA topoisomerase 2-alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsVanden Broeck A / Lamour V
Funding support France, 3 items
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, 2020-
Header (metadata) releaseMay 26, 2021-
Map releaseMay 26, 2021-
UpdateJun 2, 2021-
Current statusJun 2, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11552.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.004 / Movie #1: 0.006
Minimum - Maximum-0.027205544 - 0.034918852
Average (Standard dev.)4.416849e-05 (±0.001561242)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions296296296
Spacing296296296
CellA=B=C: 325.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z296296296
origin x/y/z0.0000.0000.000
length x/y/z325.600325.600325.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS296296296
D min/max/mean-0.0270.0350.000

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Supplemental data

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Sample components

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Entire : Etoposide-bound Entire Human Top2a

EntireName: Etoposide-bound Entire Human Top2a
Components
  • Complex: Etoposide-bound Entire Human Top2a
    • Protein or peptide: Human Top2a

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Supramolecule #1: Etoposide-bound Entire Human Top2a

SupramoleculeName: Etoposide-bound Entire Human Top2a / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Vaccinia virus Ankara / Recombinant cell: BHK21
Molecular weightTheoretical: 280 KDa

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Macromolecule #1: Human Top2a

MacromoleculeName: Human Top2a / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Vaccinia virus Ankara
SequenceString: MEVSPLQPVN ENMQVNKIKK NEDAKKRLSV ERIYQKKTQL EHILLRPDTY IGSVELVTQQ MWVYDEDVG INYREVTFVP GLYKIFDEIL VNAADNKQRD PKMSCIRVTI DPENNLISIW N NGKGIPVV EHKVEKMYVP ALIFGQLLTS SNYDDDEKKV TGGRNGYGAK ...String:
MEVSPLQPVN ENMQVNKIKK NEDAKKRLSV ERIYQKKTQL EHILLRPDTY IGSVELVTQQ MWVYDEDVG INYREVTFVP GLYKIFDEIL VNAADNKQRD PKMSCIRVTI DPENNLISIW N NGKGIPVV EHKVEKMYVP ALIFGQLLTS SNYDDDEKKV TGGRNGYGAK LCNIFSTKFT VE TASREYK KMFKQTWMDN MGRAGEMELK PFNGEDYTCI TFQPDLSKFK MQSLDKDIVA LMV RRAYDI AGSTKDVKVF LNGNKLPVKG FRSYVDMYLK DKLDETGNSL KVIHEQVNHR WEVC LTMSE KGFQQISFVN SIATSKGGRH VDYVADQIVT KLVDVVKKKN KGGVAVKAHQ VKNHM WIFV NALIENPTFD SQTKENMTLQ PKSFGSTCQL SEKFIKAAIG CGIVESILNW VKFKAQ VQL NKKCSAVKHN RIKGIPKLDD ANDAGGRNST ECTLILTEGD SAKTLAVSGL GVVGRDK YG VFPLRGKILN VREASHKQIM ENAEINNIIK IVGLQYKKNY EDEDSLKTLR YGKIMIMT D QDQDGSHIKG LLINFIHHNW PSLLRHRFLE EFITPIVKVS KNKQEMAFYS LPEFEEWKS STPNHKKWKV KYYKGLGTST SKEAKEYFAD MKRHRIQFKY SGPEDDAAIS LAFSKKQIDD RKEWLTNFM EDRRQRKLLG LPEDYLYGQT TTYLTYNDFI NKELILFSNS DNERSIPSMV D GLKPGQRK VLFTCFKRND KREVKVAQLA GSVAEMSSYH HGEMSLMMTI INLAQNFVGS NN LNLLQPI GQFGTRLHGG KDSASPRYIF TMLSSLARLL FPPKDDHTLK FLYDDNQRVE PEW YIPIIP MVLINGAEGI GTGWSCKIPN FDVREIVNNI RRLMDGEEPL PMLPSYKNFK GTIE ELAPN QYVISGEVAI LNSTTIEISE LPVRTWTQTY KEQVLEPMLN GTEKTPPLIT DYREY HTDT TVKFVVKMTE EKLAEAERVG LHKVFKLQTS LTCNSMVLFD HVGCLKKYDT VLDILR DFF ELRLKYYGLR KEWLLGMLGA ESAKLNNQAR FILEKIDGKI IIENKPKKEL IKVLIQR GY DSDPVKAWKE AQQKVPDEEE NEESDNEKET EKSDSVTDSG PTFNYLLDMP LWYLTKEK K DELCRLRNEK EQELDTLKRK SPSDLWKEDL ATFIEELEAV EAKEKQDEQV GLPGKGGKA KGKKTQMAEV LPSPRGQRVI PRITIEMKAE AEKKNKKKIK NENTEGSPQE DGVELEGLKQ RLEKKQKRE PGTKTKKQTT LAFKPIKKGK KRNPWSDSES DRSSDESNFD VPPRETEPRR A ATKTKFTM DLDSDEDFSD FDEKTDDEDF VPSDASPPKT KTSPKLSNKE LKPQKSVVSD LE ADDVKGS VPLSSSPPAT HFPDETEITN PVPKKNVTVK KTAAKSQSST STTGAKKRAA PKG TKRDPA LNSGVSQKPD PAKTKNRRKR KPSTSDDSDS NFEKIVSKAV TSKKSKGESD DFHM DFDSA VAPRAKSVRA KKPIKYLEES DEDDLF

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1908092
CTF correctionSoftware: (Name: Gctf, RELION, cryoSPARC)
Startup modelType of model: INSILICO MODEL
In silico model: An ab initio model was reconstructed using cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 36610

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Atomic model buiding 1

Initial model(PDB ID:
,
)
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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