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- PDB-8olu: Leishmania tarentolae proteasome 20S subunit in complex with 1-Be... -

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Basic information

Entry
Database: PDB / ID: 8olu
TitleLeishmania tarentolae proteasome 20S subunit in complex with 1-Benzyl-N-(3-(cyclopropylcarbamoyl)phenyl)-6-oxo-1,6-dihydropyridazine-3-carboxamide
Components
  • (Proteasome alpha ...) x 3
  • (Proteasome subunit ...) x 10
  • Proteasome beta 6 subunit, putative
KeywordsUNKNOWN FUNCTION / Proteasome 20S subunit
Function / homology
Function and homology information


proteasome core complex / proteasome core complex, beta-subunit complex / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / proteasomal protein catabolic process / proteolysis involved in protein catabolic process / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / nucleus / cytoplasm
Similarity search - Function
Proteasome subunit alpha 1 / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha5 / Proteasome subunit alpha6 / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit ...Proteasome subunit alpha 1 / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha5 / Proteasome subunit alpha6 / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome B-type subunit / Proteasome beta-type subunit profile. / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal / 4-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-VYW / Proteasome beta 6 subunit, putative / Proteasome subunit beta / Proteasome subunit alpha type / Proteasome subunit alpha type-5 / Proteasome subunit alpha type / Proteasome alpha 7 subunit, putative / Proteasome subunit beta / Proteasome subunit beta / Proteasome subunit beta ...Chem-VYW / Proteasome beta 6 subunit, putative / Proteasome subunit beta / Proteasome subunit alpha type / Proteasome subunit alpha type-5 / Proteasome subunit alpha type / Proteasome alpha 7 subunit, putative / Proteasome subunit beta / Proteasome subunit beta / Proteasome subunit beta / Proteasome subunit beta / Proteasome subunit beta / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type-1
Similarity search - Component
Biological speciesLeishmania tarentolae (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsRowland, P.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Med Chem / Year: 2023
Title: Structure-Guided Design and Synthesis of a Pyridazinone Series of Proteasome Inhibitors.
Authors: Michael G Thomas / Kate McGonagle / Paul Rowland / David A Robinson / Peter G Dodd / Isabel Camino-Díaz / Lorna Campbell / Juan Cantizani / Pablo Castañeda / Daniel Conn / Peter D Craggs / ...Authors: Michael G Thomas / Kate McGonagle / Paul Rowland / David A Robinson / Peter G Dodd / Isabel Camino-Díaz / Lorna Campbell / Juan Cantizani / Pablo Castañeda / Daniel Conn / Peter D Craggs / Darren Edwards / Liam Ferguson / Andrew Fosberry / Laura Frame / Panchali Goswami / Xiao Hu / Justyna Korczynska / Lorna MacLean / Julio Martin / Nicole Mutter / Maria Osuna-Cabello / Christy Paterson / Imanol Peña / Erika G Pinto / Caterina Pont / Jennifer Riley / Yoko Shishikura / Frederick R C Simeons / Laste Stojanovski / John Thomas / Karolina Wrobel / Robert J Young / Filip Zmuda / Fabio Zuccotto / Kevin D Read / Ian H Gilbert / Maria Marco / Timothy J Miles / Pilar Manzano / Manu De Rycker /
Abstract: There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, ...There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the proteasome. A related analogue, active against , showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.
History
DepositionMar 30, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Proteasome subunit alpha type
B: Proteasome subunit alpha type
C: Proteasome subunit alpha type
D: Proteasome subunit alpha type
E: Proteasome alpha 5 subunit, putative
F: Proteasome alpha 1 subunit, putative
G: Proteasome alpha 7 subunit, putative
H: Proteasome subunit beta
I: Proteasome subunit beta
J: Proteasome subunit beta
K: Proteasome subunit beta
L: Proteasome subunit beta
M: Proteasome beta 6 subunit, putative
N: Proteasome subunit beta
O: Proteasome subunit alpha type
P: Proteasome subunit alpha type
Q: Proteasome subunit alpha type
R: Proteasome subunit alpha type
S: Proteasome alpha 5 subunit, putative
T: Proteasome alpha 1 subunit, putative
U: Proteasome alpha 7 subunit, putative
V: Proteasome subunit beta
W: Proteasome subunit beta
X: Proteasome subunit beta
Y: Proteasome subunit beta
Z: Proteasome subunit beta
a: Proteasome beta 6 subunit, putative
b: Proteasome subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)848,36530
Polymers847,58828
Non-polymers7772
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area123330 Å2
ΔGint-422 kcal/mol
Surface area202550 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
22
33
44
55
66
77
88
99
1010
1111
1212
1313
1414
/ NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14

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Components

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Proteasome subunit ... , 10 types, 20 molecules AOBPCQDRHVIWJXKYLZNb

#1: Protein Proteasome subunit alpha type


Mass: 27178.107 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KZP5
#2: Protein Proteasome subunit alpha type


Mass: 25179.559 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KGL4
#3: Protein Proteasome subunit alpha type


Mass: 32321.438 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KBV2
#4: Protein Proteasome subunit alpha type


Mass: 27821.605 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KXA2
#8: Protein Proteasome subunit beta


Mass: 30280.010 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KBR2
#9: Protein Proteasome subunit beta


Mass: 27603.570 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KUX2
#10: Protein Proteasome subunit beta


Mass: 22470.887 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KQX8
#11: Protein Proteasome subunit beta


Mass: 23065.291 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KTY7
#12: Protein Proteasome subunit beta


Mass: 33576.738 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KW57
#14: Protein Proteasome subunit beta


Mass: 24737.232 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KSC5

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Proteasome alpha ... , 3 types, 6 molecules ESFTGU

#5: Protein Proteasome alpha 5 subunit, putative


Mass: 38312.316 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KG82
#6: Protein Proteasome alpha 1 subunit, putative


Mass: 47978.633 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640L0A1
#7: Protein Proteasome alpha 7 subunit, putative


Mass: 25591.826 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640KJI7

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Protein / Non-polymers , 2 types, 4 molecules Ma

#13: Protein Proteasome beta 6 subunit, putative


Mass: 37676.910 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Leishmania tarentolae (eukaryote) / References: UniProt: A0A640K9U9
#15: Chemical ChemComp-VYW / ~{N}-[3-(cyclopropylcarbamoyl)phenyl]-6-oxidanylidene-1-(phenylmethyl)pyridazine-3-carboxamide


Mass: 388.419 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C22H20N4O3

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Leishmania tarentolae proteasome 20S subunit in complex with 1-Benzyl-N-(3-(cyclopropylcarbamoyl)phenyl)-6-oxo-1,6-dihydropyridazine-3-carboxamide
Type: COMPLEX / Entity ID: #1-#11, #13-#14 / Source: NATURAL
Source (natural)Organism: Leishmania tarentolae (eukaryote)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3400 nm / Nominal defocus min: 1800 nm
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
9REFMAC5.8.0349model refinement
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94595 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingDetails: Unpublished model / Source name: Other / Type: experimental model
RefinementResolution: 2.59→186.18 Å / Cor.coef. Fo:Fc: 0.895 / SU B: 6.111 / SU ML: 0.122 / ESU R: 0.235
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.24904 --
obs0.24904 564206 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 27.864 Å2
Baniso -1Baniso -2Baniso -3
1-0.24 Å2-0.48 Å2-0 Å2
2---0.13 Å2-0 Å2
3----0.11 Å2
Refinement stepCycle: 1 / Total: 48930
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0160.01249860
ELECTRON MICROSCOPYr_bond_other_d0.0010.01645300
ELECTRON MICROSCOPYr_angle_refined_deg2.1281.6467458
ELECTRON MICROSCOPYr_angle_other_deg0.7411.556105490
ELECTRON MICROSCOPYr_dihedral_angle_1_deg8.00356286
ELECTRON MICROSCOPYr_dihedral_angle_2_deg17.2810344
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.918108468
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0860.27516
ELECTRON MICROSCOPYr_gen_planes_refined0.0110.0257574
ELECTRON MICROSCOPYr_gen_planes_other0.0010.029946
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it3.6442.28125238
ELECTRON MICROSCOPYr_mcbond_other3.6442.28125238
ELECTRON MICROSCOPYr_mcangle_it5.813.41431492
ELECTRON MICROSCOPYr_mcangle_other5.8113.41431493
ELECTRON MICROSCOPYr_scbond_it5.883.25424622
ELECTRON MICROSCOPYr_scbond_other5.883.25524623
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other9.5354.49335961
ELECTRON MICROSCOPYr_long_range_B_refined11.78927.23956406
ELECTRON MICROSCOPYr_long_range_B_other11.78927.2456407
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY

Ens-IDDom-IDAuth asym-IDNumberTypeRms dev position (Å)Weight position
1010J1557tight positional00.05
1111K1612tight positional00.05
1212L1579tight positional00.05
1313M1702tight positional00.05
1414N1732tight positional00.05
11A1857tight thermal0.170.5
22B1754tight thermal0.180.5
33C2150tight thermal0.250.5
44D1840tight thermal0.470.5
55E1756tight thermal0.260.5
66F1819tight thermal0.240.5
77G1714tight thermal0.210.5
88H1705tight thermal0.150.5
99I1659tight thermal0.420.5
1010J1557tight thermal0.210.5
1111K1612tight thermal0.170.5
1212L1579tight thermal0.140.5
1313M1702tight thermal0.180.5
1414N1732tight thermal0.140.5
LS refinement shellResolution: 2.589→2.656 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.524 41750 -
obs--100 %

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