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- EMDB-11554: Cryo-EM structure of the entire Human topoisomerase II alpha in S... -

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Entry
Database: EMDB / ID: EMD-11554
TitleCryo-EM structure of the entire Human topoisomerase II alpha in State 2
Map data
Sample
  • Complex: Etoposide-bound Entire Human Top2a in state 2
    • Complex: DNA topoisomerase 2-alpha
      • Protein or peptide: DNA topoisomerase 2-alpha
    • Complex: DNA
      • DNA: DNA (5'-D(*CP*GP*CP*GP*CP*AP*TP*CP*GP*TP*CP*AP*TP*CP*CP*TP*C)-3')
      • DNA: DNA (5'-D(*GP*AP*GP*GP*AP*TP*GP*AP*CP*GP*AP*TP*G)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: (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
KeywordsHuman Topoisomerase / Etoposide / DNA / ISOMERASE
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 / embryonic cleavage / DNA ligation / 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 / embryonic cleavage / DNA ligation / 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 / protein kinase C binding / ubiquitin binding / male germ cell nucleus / chromosome segregation / regulation of circadian rhythm / rhythmic process / ribonucleoprotein complex / positive regulation of apoptotic process / 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 / 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
DNA topoisomerase 2-alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.4 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-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zy8
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11554.png

Downloads & links

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Map

FileDownload / File: emd_11554.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.507 Å
Density
Contour LevelBy AUTHOR: 0.285 / Movie #1: 0.3
Minimum - Maximum-0.20996946 - 1.0480946
Average (Standard dev.)0.0048406427 (±0.058191646)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 325.512 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.5071.5071.507
M x/y/z216216216
origin x/y/z0.0000.0000.000
length x/y/z325.512325.512325.512
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS216216216
D min/max/mean-0.2101.0480.005

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Supplemental data

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Mask #1

Fileemd_11554_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11554_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11554_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Etoposide-bound Entire Human Top2a in state 2

EntireName: Etoposide-bound Entire Human Top2a in state 2
Components
  • Complex: Etoposide-bound Entire Human Top2a in state 2
    • Complex: DNA topoisomerase 2-alpha
      • Protein or peptide: DNA topoisomerase 2-alpha
    • Complex: DNA
      • DNA: DNA (5'-D(*CP*GP*CP*GP*CP*AP*TP*CP*GP*TP*CP*AP*TP*CP*CP*TP*C)-3')
      • DNA: DNA (5'-D(*GP*AP*GP*GP*AP*TP*GP*AP*CP*GP*AP*TP*G)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: (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

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Supramolecule #1: Etoposide-bound Entire Human Top2a in state 2

SupramoleculeName: Etoposide-bound Entire Human Top2a in state 2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 295 KDa

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Supramolecule #2: DNA topoisomerase 2-alpha

SupramoleculeName: DNA topoisomerase 2-alpha / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA topoisomerase 2-alpha

MacromoleculeName: DNA topoisomerase 2-alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA topoisomerase (ATP-hydrolysing)
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 174.654406 KDa
Recombinant expressionOrganism: Vaccinia virus Ankara
SequenceString: MEVSPLQPVN ENMQVNKIKK NEDAKKRLSV ERIYQKKTQL EHILLRPDTY IGSVELVTQQ MWVYDEDVGI NYREVTFVPG LYKIFDEIL VNAADNKQRD PKMSCIRVTI DPENNLISIW NNGKGIPVVE HKVEKMYVPA LIFGQLLTSS NYDDDEKKVT G GRNGYGAK ...String:
MEVSPLQPVN ENMQVNKIKK NEDAKKRLSV ERIYQKKTQL EHILLRPDTY IGSVELVTQQ MWVYDEDVGI NYREVTFVPG LYKIFDEIL VNAADNKQRD PKMSCIRVTI DPENNLISIW NNGKGIPVVE HKVEKMYVPA LIFGQLLTSS NYDDDEKKVT G GRNGYGAK LCNIFSTKFT VETASREYKK MFKQTWMDNM GRAGEMELKP FNGEDYTCIT FQPDLSKFKM QSLDKDIVAL MV RRAYDIA GSTKDVKVFL NGNKLPVKGF RSYVDMYLKD KLDETGNSLK VIHEQVNHRW EVCLTMSEKG FQQISFVNSI ATS KGGRHV DYVADQIVTK LVDVVKKKNK GGVAVKAHQV KNHMWIFVNA LIENPTFDSQ TKENMTLQPK SFGSTCQLSE KFIK AAIGC GIVESILNWV KFKAQVQLNK KCSAVKHNRI KGIPKLDDAN DAGGRNSTEC TLILTEGDSA KTLAVSGLGV VGRDK YGVF PLRGKILNVR EASHKQIMEN AEINNIIKIV GLQYKKNYED EDSLKTLRYG KIMIMTDQDQ DGSHIKGLLI NFIHHN WPS LLRHRFLEEF ITPIVKVSKN KQEMAFYSLP EFEEWKSSTP NHKKWKVKYY KGLGTSTSKE AKEYFADMKR HRIQFKY SG PEDDAAISLA FSKKQIDDRK EWLTNFMEDR RQRKLLGLPE DYLYGQTTTY LTYNDFINKE LILFSNSDNE RSIPSMVD G LKPGQRKVLF TCFKRNDKRE VKVAQLAGSV AEMSSYHHGE MSLMMTIINL AQNFVGSNNL NLLQPIGQFG TRLHGGKDS ASPRYIFTML SSLARLLFPP KDDHTLKFLY DDNQRVEPEW YIPIIPMVLI NGAEGIGTGW SCKIPNFDVR EIVNNIRRLM DGEEPLPML PSYKNFKGTI EELAPNQYVI SGEVAILNST TIEISELPVR TWTQTYKEQV LEPMLNGTEK TPPLITDYRE Y HTDTTVKF VVKMTEEKLA EAERVGLHKV FKLQTSLTCN SMVLFDHVGC LKKYDTVLDI LRDFFELRLK YYGLRKEWLL GM LGAESAK LNNQARFILE KIDGKIIIEN KPKKELIKVL IQRGYDSDPV KAWKEAQQKV PDEEENEESD NEKETEKSDS VTD SGPTFN YLLDMPLWYL TKEKKDELCR LRNEKEQELD TLKRKSPSDL WKEDLATFIE ELEAVEAKEK QDEQVGLPGK GGKA KGKKT QMAEVLPSPR GQRVIPRITI EMKAEAEKKN KKKIKNENTE GSPQEDGVEL EGLKQRLEKK QKREPGTKTK KQTTL AFKP IKKGKKRNPW SDSESDRSSD ESNFDVPPRE TEPRRAATKT KFTMDLDSDE DFSDFDEKTD DEDFVPSDAS PPKTKT SPK LSNKELKPQK SVVSDLEADD VKGSVPLSSS PPATHFPDET EITNPVPKKN VTVKKTAAKS QSSTSTTGAK KRAAPKG TK RDPALNSGVS QKPDPAKTKN RRKRKPSTSD DSDSNFEKIV SKAVTSKKSK GESDDFHMDF DSAVAPRAKS VRAKKPIK Y LEESDEDDLF

UniProtKB: DNA topoisomerase 2-alpha

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Macromolecule #2: DNA (5'-D(*CP*GP*CP*GP*CP*AP*TP*CP*GP*TP*CP*AP*TP*CP*CP*TP*C)-3')

MacromoleculeName: DNA (5'-D(*CP*GP*CP*GP*CP*AP*TP*CP*GP*TP*CP*AP*TP*CP*CP*TP*C)-3')
type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 5.099298 KDa
SequenceString:
(DC)(DG)(DC)(DG)(DC)(DA)(DT)(DC)(DG)(DT) (DC)(DA)(DT)(DC)(DC)(DT)(DC)

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Macromolecule #3: DNA (5'-D(*GP*AP*GP*GP*AP*TP*GP*AP*CP*GP*AP*TP*G)-3')

MacromoleculeName: DNA (5'-D(*GP*AP*GP*GP*AP*TP*GP*AP*CP*GP*AP*TP*G)-3') / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 4.080671 KDa
SequenceString:
(DG)(DA)(DG)(DG)(DA)(DT)(DG)(DA)(DC)(DG) (DA)(DT)(DG)

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Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 2 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #5: (5S,5aR,8aR,9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,...

MacromoleculeName: (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
type: ligand / ID: 5 / Number of copies: 2 / Formula: EVP
Molecular weightTheoretical: 588.557 Da

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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
Startup model#0 - Type of model: INSILICO MODEL
#0 - In silico model: An ab initio model was reconstructed using cryoSPARC
#1 - Type of model: INSILICO MODEL
#1 - In silico model: An ab initio model was reconstructed using cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3) / Number images used: 13420
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5gwk

source_name: PDB, initial_model_type: experimental model

1zxm

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6zy8:
Cryo-EM structure of the entire Human topoisomerase II alpha in State 2

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