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- EMDB-36116: Cryo-EM structure of the African swine fever virus topoisomerase ... -

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Entry
Database: EMDB / ID: EMD-36116
TitleCryo-EM structure of the African swine fever virus topoisomerase 2 complexed with Cut02aDNA and etoposide (EDI-1)
Map data
Sample
  • Complex: African swine fever virus topoisomerase 2 complexed with Cut02aDNA and etoposide
    • Protein or peptide: DNA topoisomerase 2
    • DNA: DNA (5'-D(*GP*AP*GP*GP*TP*AP*TP*GP*TP*AP*GP*GP*C)-3')
    • DNA: DNA (5'-D(*GP*GP*CP*CP*GP*CP*CP*TP*AP*CP*AP*TP*AP*CP*CP*TP*C)-3')
  • Ligand: MAGNESIUM ION
  • 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
KeywordsTopoisomerase / ASFV / inhibitor / VIRAL PROTEIN / ISOMERASE-DNA complex
Function / homology
Function and homology information


sister chromatid segregation / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / DNA binding / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / DNA topoisomerase II, eukaryotic-type / : / 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 ...C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / DNA topoisomerase II, eukaryotic-type / : / 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 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsChang C-W / Tsai M-D
Funding support Taiwan, 1 items
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 5, 2023-
Header (metadata) releaseFeb 7, 2024-
Map releaseFeb 7, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36116.png

Downloads & links

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Map

FileDownload / File: emd_36116.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 432 pix.
= 358.56 Å		0.83 Å/pix.
x 432 pix.
= 358.56 Å		0.83 Å/pix.
x 432 pix.
= 358.56 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.148
Minimum - Maximum-0.34060603 - 1.1430036
Average (Standard dev.)0.0011442951 (±0.023103124)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 358.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_36116_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_36116_half_map_2.map
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Sample components

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Entire : African swine fever virus topoisomerase 2 complexed with Cut02aDN...

EntireName: African swine fever virus topoisomerase 2 complexed with Cut02aDNA and etoposide
Components
  • Complex: African swine fever virus topoisomerase 2 complexed with Cut02aDNA and etoposide
    • Protein or peptide: DNA topoisomerase 2
    • DNA: DNA (5'-D(*GP*AP*GP*GP*TP*AP*TP*GP*TP*AP*GP*GP*C)-3')
    • DNA: DNA (5'-D(*GP*GP*CP*CP*GP*CP*CP*TP*AP*CP*AP*TP*AP*CP*CP*TP*C)-3')
  • Ligand: MAGNESIUM ION
  • 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: African swine fever virus topoisomerase 2 complexed with Cut02aDN...

SupramoleculeName: African swine fever virus topoisomerase 2 complexed with Cut02aDNA and etoposide
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: African swine fever virus

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Macromolecule #1: DNA topoisomerase 2

MacromoleculeName: DNA topoisomerase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: African swine fever virus
Molecular weightTheoretical: 136.422578 KDa
Recombinant expressionOrganism: Saccharomyces (fungus)
SequenceString: MEAFEISDFK EHAKKKSMWA GALNKVTISG LMGVFTEDED LMALPIHRDH CPALLKIFDE LIVNATDHER ACHSKTKKVT YIKISFDKG VFSCENDGPG IPIAKHEQAS LIAKRDVYVP EVASCFFLAG TNINKAKDCI KGGTNGVGLK LAMVHSQWAI L TTADGAQK ...String:
MEAFEISDFK EHAKKKSMWA GALNKVTISG LMGVFTEDED LMALPIHRDH CPALLKIFDE LIVNATDHER ACHSKTKKVT YIKISFDKG VFSCENDGPG IPIAKHEQAS LIAKRDVYVP EVASCFFLAG TNINKAKDCI KGGTNGVGLK LAMVHSQWAI L TTADGAQK YVQQINQRLD IIEPPTITPS REMFTRIELM PVYQELGYAE PLSETEQADL SAWIYLRACQ CAAYVGKGTT IY YNDKPCR TGSVMALAKM YTLLSAPNST IHTATIKADA KPYSLHPLQV AAVVSPKFKK FEHVSIINGV NCVKGEHVTF LKK TINEMV IKKFQQTIKD KNRKTTLRDS CSNIFVVIVG SIPGIEWTGQ RKDELSIAEN VFKTHYSIPS SFLTSMTRSI VDIL LQSIS KKDNHKQVDV DKYTRARNAG GKRAQDCMLL AAEGDSALSL LRTGLTLGKS NPSGPSFDFC GMISLGGVIM NACKK VTNI TTDSGETIMV RNEQLTNNKV LQGIVQVLGL DFNCHYKTQE ERAKLRYGCI VACVDQDLDG CGKILGLLLA YFHLFW PQL IIHGFVKRLL TPLIRVYEKG KTMPVEFYYE QEFDAWAKKQ TSLVNHTVKY YKGLAAHDTH EVKSMFKHFD NMVYTFT LD DSAKELFHIY FGGESELRKR ELCTGVVPLT ETQTQSIHSV RRIPCSLHLQ VDTKAYKLDA IERQIPNFLD GMTRARRK I LAGGVKCFAS NNRERKVFQF GGYVADHMFY HHGDMSLNTS IIKAAQYYPG SSHLYPVFIG IGSFGSRHLG GKDAGSPRY ISVQLASEFI KTMFPAEDSW LLPYVFEDGQ RAEPEYYVPV LPLAIMEYGA NPSEGWKYTT WARQLEDILA LVRAYVDKDN PKHELLHYA IKHKITILPL RPSNYNFKGH LKRFGQYYYS YGTYDISEQR NIITITELPL RVPTVAYIES IKKSSNRMTF I EEIIDYSS SETIEILVKL KPNSLNRIVE EFKETEEQDS IENFLRLRNC LHSHLNFVKP KGGIIEFNSY YEILYAWLPY RR ELYQKRL MREHAVLKLR IIMETAIVRY INESAELNLS HYEDEKEASR ILSEHGFPPL NHTLIISPEF ASIEELNQKA LQG CYTYIL SLQARELLIA AKTRRVEKIK KMQARLDKVE QLLQESPFPG ASVWLEEIDA VEKAIIKGRN TQWKFHHHHH H

UniProtKB: DNA topoisomerase 2

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

MacromoleculeName: DNA (5'-D(*GP*AP*GP*GP*TP*AP*TP*GP*TP*AP*GP*GP*C)-3') / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 4.071657 KDa
SequenceString:
(DG)(DA)(DG)(DG)(DT)(DA)(DT)(DG)(DT)(DA) (DG)(DG)(DC)

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

MacromoleculeName: DNA (5'-D(*GP*GP*CP*CP*GP*CP*CP*TP*AP*CP*AP*TP*AP*CP*CP*TP*C)-3')
type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 5.108312 KDa
SequenceString:
(DG)(DG)(DC)(DC)(DG)(DC)(DC)(DT)(DA)(DC) (DA)(DT)(DA)(DC)(DC)(DT)(DC)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.3 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 11786 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: ASFV Topoisomerase in apo conformer IIa
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 632007
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainDetailsPDB ID
source_name: Other, initial_model_type: experimental modelapo-AsfvTop2 conformer IIa
source_name: PDB, initial_model_type: experimental model

6zy7

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8j9v:
Cryo-EM structure of the African swine fever virus topoisomerase 2 complexed with Cut02aDNA and etoposide (EDI-1)

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