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- EMDB-36473: Cryo-EM structure of the full-length African swine fever virus to... -

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Entry
Database: EMDB / ID: EMD-36473
TitleCryo-EM structure of the full-length African swine fever virus topoisomerase 2 complexed with Cut02aDNA and etoposide
Map data
Sample
  • Complex: ASFV Topoisomerase 2 complexed with Cut02aDNA and etoposide
    • Protein or peptide: African swine fever virus type 2 topoisomerase
KeywordsTopoisomerase / ASFV / inhibitor / VIRAL PROTEIN
Biological speciesAfrican swine fever virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsChang CW / Tsai MD
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
DepositionJun 11, 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_36473.png

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Map

FileDownload / File: emd_36473.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.126
Minimum - Maximum-0.30203307 - 0.9369518
Average (Standard dev.)0.0020035258 (±0.022392534)
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: #1

Fileemd_36473_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Half map: #2

Fileemd_36473_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : ASFV Topoisomerase 2 complexed with Cut02aDNA and etoposide

EntireName: ASFV Topoisomerase 2 complexed with Cut02aDNA and etoposide
Components
  • Complex: ASFV Topoisomerase 2 complexed with Cut02aDNA and etoposide
    • Protein or peptide: African swine fever virus type 2 topoisomerase

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Supramolecule #1: ASFV Topoisomerase 2 complexed with Cut02aDNA and etoposide

SupramoleculeName: ASFV Topoisomerase 2 complexed with Cut02aDNA and etoposide
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: African swine fever virus

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Macromolecule #1: African swine fever virus type 2 topoisomerase

MacromoleculeName: African swine fever virus type 2 topoisomerase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: African swine fever virus
Recombinant expressionOrganism: Saccharomyces (fungus)
SequenceString: MEAFEISDFK EHAKKKSMWA GALNKVTISG LMGVFTEDED LMALPIHRDH CPALLKIFDE LIVNATDHE RACHSKTKKV TYIKISFDKG VFSCENDGPG IPIAKHEQAS LIAKRDVYVP E VASCFFLA GTNINKAKDC IKGGTNGVGL KLAMVHSQWA ILTTADGAQK ...String:
MEAFEISDFK EHAKKKSMWA GALNKVTISG LMGVFTEDED LMALPIHRDH CPALLKIFDE LIVNATDHE RACHSKTKKV TYIKISFDKG VFSCENDGPG IPIAKHEQAS LIAKRDVYVP E VASCFFLA GTNINKAKDC IKGGTNGVGL KLAMVHSQWA ILTTADGAQK YVQQINQRLD II EPPTITP SREMFTRIEL MPVYQELGYA EPLSETEQAD LSAWIYLRAC QCAAYVGKGT TIY YNDKPC RTGSVMALAK MYTLLSAPNS TIHTATIKAD AKPYSLHPLQ VAAVVSPKFK KFEH VSIIN GVNCVKGEHV TFLKKTINEM VIKKFQQTIK DKNRKTTLRD SCSNIFVVIV GSIPG IEWT GQRKDELSIA ENVFKTHYSI PSSFLTSMTR SIVDILLQSI SKKDNHKQVD VDKYTR ARN AGGKRAQDCM LLAAEGDSAL SLLRTGLTLG KSNPSGPSFD FCGMISLGGV IMNACKK VT NITTDSGETI MVRNEQLTNN KVLQGIVQVL GLDFNCHYKT QEERAKLRYG CIVACVDQ D LDGCGKILGL LLAYFHLFWP QLIIHGFVKR LLTPLIRVYE KGKTMPVEFY YEQEFDAWA KKQTSLVNHT VKYYKGLAAH DTHEVKSMFK HFDNMVYTFT LDDSAKELFH IYFGGESELR KRELCTGVV PLTETQTQSI HSVRRIPCSL HLQVDTKAYK LDAIERQIPN FLDGMTRARR K ILAGGVKC FASNNRERKV FQFGGYVADH MFYHHGDMSL NTSIIKAAQY YPGSSHLYPV FI GIGSFGS RHLGGKDAGS PRYISVQLAS EFIKTMFPAE DSWLLPYVFE DGQRAEPEYY VPV LPLAIM EYGANPSEGW KYTTWARQLE DILALVRAYV DKDNPKHELL HYAIKHKITI LPLR PSNYN FKGHLKRFGQ YYYSYGTYDI SEQRNIITIT ELPLRVPTVA YIESIKKSSN RMTFI EEII DYSSSETIEI LVKLKPNSLN RIVEEFKETE EQDSIENFLR LRNCLHSHLN FVKPKG GII EFNSYYEILY AWLPYRRELY QKRLMREHAV LKLRIIMETA IVRYINESAE LNLSHYE DE KEASRILSEH GFPPLNHTLI ISPEFASIEE LNQKALQGCY TYILSLQARE LLIAAKTR R VEKIKKMQAR LDKVEQLLQE SPFPGASVWL EEIDAVEKAI IKGRNTQWKF H

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Sample stageCooling holder cryogen: NITROGEN
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 80745
FSC plot (resolution estimation)

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