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- EMDB-29930: T. cruzi topoisomerase II alpha bound to dsDNA and the covalent i... -

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Basic information

Entry
Database: EMDB / ID: EMD-29930
TitleT. cruzi topoisomerase II alpha bound to dsDNA and the covalent inhibitor CT1
Map dataFinal refined and sharpened map from NU refinement.
Sample
  • Complex: Topoisomerase II alpha bound to dsDNA and inhibitor CT1
    • Complex: Topoisomerase II alpha
      • Complex: dsDNADNA
        • DNA: DNA (28-MER)
      • Protein or peptide: DNA topoisomerase 2Topoisomerase
  • Ligand: 2-{3-[(Z)-iminomethyl]-1H-1,2,4-triazol-1-yl}-1-{(3M)-3-[2-(trifluoromethyl)phenyl]-6H-pyrrolo[3,4-b]pyridin-6-yl}ethan-1-one
KeywordsTopoisomerase / DNA binding protein / Topoisomerase inhibitor / ISOMERASE
Function / homology
Function and homology information


DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / DNA binding / ATP binding / metal ion binding
Similarity search - Function
DNA topoisomerase 2, TOPRIM domain / DNA topoisomerase II, eukaryotic-type / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / 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 topoisomerase 2, TOPRIM domain / DNA topoisomerase II, eukaryotic-type / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / 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 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
Biological speciesTrypanosoma cruzi (eukaryote) / Escherichia coli (E. coli) / Trypanosoma cruzi strain CL Brener (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsSchenk A / Deniston C / Noeske J
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust219639/Z/19/Z United Kingdom
Wellcome Trust108517/Z/15/Z United Kingdom
CitationJournal: Science / Year: 2023
Title: Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections.
Authors: Srinivasa P S Rao / Matthew K Gould / Jonas Noeske / Manuel Saldivia / Rajiv S Jumani / Pearly S Ng / Olivier René / Yen-Liang Chen / Marcel Kaiser / Ryan Ritchie / Amanda Fortes Francisco ...Authors: Srinivasa P S Rao / Matthew K Gould / Jonas Noeske / Manuel Saldivia / Rajiv S Jumani / Pearly S Ng / Olivier René / Yen-Liang Chen / Marcel Kaiser / Ryan Ritchie / Amanda Fortes Francisco / Nila Johnson / Debjani Patra / Harry Cheung / Colin Deniston / Andreas D Schenk / Wilian A Cortopassi / Remo S Schmidt / Natalie Wiedemar / Bryanna Thomas / Rima Palkar / Nahdiyah A Ghafar / Vanessa Manoharan / Catherine Luu / Jonathan E Gable / Kah Fei Wan / Elmarie Myburgh / Jeremy C Mottram / Whitney Barnes / John Walker / Charles Wartchow / Natasha Aziz / Colin Osborne / Juergen Wagner / Christopher Sarko / John M Kelly / Ujjini H Manjunatha / Pascal Mäser / Jan Jiricek / Suresh B Lakshminarayana / Michael P Barrett / Thierry T Diagana /
Abstract: Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are ...Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.
History
DepositionMar 1, 2023-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateJul 12, 2023-
Current statusJul 12, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29930.png

Downloads & links

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Map

FileDownload / File: emd_29930.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal refined and sharpened map from NU refinement.
Voxel sizeX=Y=Z: 0.845 Å
Density
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-2.2801254 - 4.1476197
Average (Standard dev.)-0.0003583988 (±0.06518587)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 324.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29930_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Final half map from NU refinement.

Fileemd_29930_half_map_1.map
AnnotationFinal half map from NU refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Final half map from NU refinement.

Fileemd_29930_half_map_2.map
AnnotationFinal half map from NU refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Topoisomerase II alpha bound to dsDNA and inhibitor CT1

EntireName: Topoisomerase II alpha bound to dsDNA and inhibitor CT1
Components
  • Complex: Topoisomerase II alpha bound to dsDNA and inhibitor CT1
    • Complex: Topoisomerase II alpha
      • Complex: dsDNADNA
        • DNA: DNA (28-MER)
      • Protein or peptide: DNA topoisomerase 2Topoisomerase
  • Ligand: 2-{3-[(Z)-iminomethyl]-1H-1,2,4-triazol-1-yl}-1-{(3M)-3-[2-(trifluoromethyl)phenyl]-6H-pyrrolo[3,4-b]pyridin-6-yl}ethan-1-one

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Supramolecule #1: Topoisomerase II alpha bound to dsDNA and inhibitor CT1

SupramoleculeName: Topoisomerase II alpha bound to dsDNA and inhibitor CT1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Trypanosoma cruzi (eukaryote)

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Supramolecule #2: Topoisomerase II alpha

SupramoleculeName: Topoisomerase II alpha / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Trypanosoma cruzi (eukaryote)

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Supramolecule #3: dsDNA

SupramoleculeName: dsDNA / type: complex / ID: 3 / Parent: 2 / Macromolecule list: #2
Details: DNA sequence: GG GAT AAC AAT GAG CTC ATT GTT ATC CC
Source (natural)Organism: Escherichia coli (E. coli)

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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: Trypanosoma cruzi strain CL Brener (eukaryote)
Molecular weightTheoretical: 89.322438 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GRNADRKQIL GIPKLDDANE AGGKYSHRCT LILTEGDSAK ALCTAGLAVK DRDYFGVFPL RGKPLNVRDA TLKKVMACAE FQAVSKIMG LDIRQKYSGV ERLRYGHLMI MSDQDHDGSH IKGLIINMIH HYWPDLIKTP GFLQQFITPI VKARKKGRSD G DDRAISFF ...String:
GRNADRKQIL GIPKLDDANE AGGKYSHRCT LILTEGDSAK ALCTAGLAVK DRDYFGVFPL RGKPLNVRDA TLKKVMACAE FQAVSKIMG LDIRQKYSGV ERLRYGHLMI MSDQDHDGSH IKGLIINMIH HYWPDLIKTP GFLQQFITPI VKARKKGRSD G DDRAISFF SMPDYFEWKN AIGDGIRNYE IRYYKGLGTS GAKEGREYFE NIDRHRLDFV HEDATDDARI VMAFAKDKVE ER KHWITQF KANTNVNESM NYNVRTVRYS EFVDKELILF SVADCERSIP SVIDGLKPGQ RKIIFSSFKR RLTRSIKVVQ LAG YVSEHA AYHHGEQSLV QTIVGLAQNF VGSNNVPLLQ QDGQFGTRLQ GGKDHAAGRY IFTRLTNIAR YIYHPSDDFV VDYK DDDGL SVEPFYYVPV IPMVLVNGTS GIGTGFATNI PNYSPLEVID NLMRLLRGEE VQPMKPWYFG FAGTIEEKEK GKFVS TGCA NVRPDGVVQI TELPIGTWTQ GYKKFLEELR EKEVVVQYRE HNTDVTVDFE VFLHPEVLHH WVAQGCVEER LQLREY IHA TNIIAFDREG QITKYRDAEA VLKEFYLVRL EYYAKRRDFL IGDLRSVASK LENMVRFVTE VVDGRLIVTR RRKKELL EE LRQRGYAPFP LQQKKKVSST TIQQGEEEGA ADATHATAED VFLVLQPAVD EGGDEDNQET PEMRRAARDY DYLLGMRL W NLTAEMIARL QSQLQKARDE LAALEKRTPK DLWAEDLNQL RPRIENLFEE RAKEIASI

UniProtKB: DNA topoisomerase 2

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Macromolecule #2: DNA (28-MER)

MacromoleculeName: DNA (28-MER) / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 8.604566 KDa
SequenceString:
(DG)(DG)(DG)(DA)(DT)(DA)(DA)(DC)(DA)(DA) (DT)(DG)(DA)(DG)(DC)(DT)(DC)(DA)(DT)(DT) (DG)(DT)(DT)(DA)(DT)(DC)(DC)(DC)

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Macromolecule #3: 2-{3-[(Z)-iminomethyl]-1H-1,2,4-triazol-1-yl}-1-{(3M)-3-[2-(trifl...

MacromoleculeName: 2-{3-[(Z)-iminomethyl]-1H-1,2,4-triazol-1-yl}-1-{(3M)-3-[2-(trifluoromethyl)phenyl]-6H-pyrrolo[3,4-b]pyridin-6-yl}ethan-1-one
type: ligand / ID: 3 / Number of copies: 2 / Formula: YWX
Molecular weightTheoretical: 398.341 Da
Chemical component information

ChemComp-YWX:
2-{3-[(Z)-iminomethyl]-1H-1,2,4-triazol-1-yl}-1-{(3M)-3-[2-(trifluoromethyl)phenyl]-6H-pyrrolo[3,4-b]pyridin-6-yl}ethan-1-one

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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

Concentration0.5 mg/mL
BufferpH: 7.3
Component:
ConcentrationNameFormula
0.02 MHEPES
0.1 Mpotassium chlorideKCl
0.003 Mmagnesium chlorideMgCl2
0.001 MTCEP
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 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: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: FEI FALCON IV (4k x 4k) / #0 - Digitization - Dimensions - Width: 4096 pixel / #0 - Digitization - Dimensions - Height: 4096 pixel / #0 - Number grids imaged: 1 / #0 - Number real images: 3014 / #0 - Average exposure time: 2.8 sec. / #0 - Average electron dose: 50.0 e/Å2
#0 - Details: Micrographs collected at 30 degrees tilt using a Falcon 4i camera.
#1 - Image recording ID: 2 / #1 - Film or detector model: FEI FALCON IV (4k x 4k) / #1 - Digitization - Dimensions - Width: 4096 pixel / #1 - Digitization - Dimensions - Height: 4096 pixel / #1 - Number grids imaged: 1 / #1 - Number real images: 2220 / #1 - Average exposure time: 2.98 sec. / #1 - Average electron dose: 50.0 e/Å2
#1 - Details: Micrographs collected at 30 degrees tilt using a Falcon 4 camera.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1659398
Details: 955991 particles for tilted data 703407 particles for untilted data
Startup modelType of model: INSILICO MODEL
In silico model: Initial model generated using ab-inito reconstruction in cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 179787
DetailsUntilted and tilted data merged
Image recording ID1
FSC plot (resolution estimation)

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