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- PDB-6tu3: Rat 20S proteasome -

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Basic information

Entry
Database: PDB / ID: 6tu3
TitleRat 20S proteasome
Components
  • (Proteasome subunit alpha type- ...) x 7
  • (Proteasome subunit beta type- ...) x 7
KeywordsHYDROLASE / PROTEASOME
Function / homology
Function and homology information


Cross-presentation of soluble exogenous antigens (endosomes) / SCF-beta-TrCP mediated degradation of Emi1 / Regulation of ornithine decarboxylase (ODC) / Assembly of the pre-replicative complex / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / Cdc20:Phospho-APC/C mediated degradation of Cyclin A ...Cross-presentation of soluble exogenous antigens (endosomes) / SCF-beta-TrCP mediated degradation of Emi1 / Regulation of ornithine decarboxylase (ODC) / Assembly of the pre-replicative complex / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / SCF(Skp2)-mediated degradation of p27/p21 / Autodegradation of the E3 ubiquitin ligase COP1 / Asymmetric localization of PCP proteins / Degradation of DVL / Hedgehog ligand biogenesis / Dectin-1 mediated noncanonical NF-kB signaling / Degradation of GLI1 by the proteasome / Hedgehog 'on' state / TNFR2 non-canonical NF-kB pathway / NIK-->noncanonical NF-kB signaling / UCH proteinases / CDK-mediated phosphorylation and removal of Cdc6 / G2/M Checkpoints / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Ubiquitin-dependent degradation of Cyclin D / The role of GTSE1 in G2/M progression after G2 checkpoint / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Regulation of RUNX3 expression and activity / GLI3 is processed to GLI3R by the proteasome / Activation of NF-kappaB in B cells / Degradation of beta-catenin by the destruction complex / Degradation of AXIN / Regulation of RAS by GAPs / Orc1 removal from chromatin / Neddylation / AUF1 (hnRNP D0) binds and destabilizes mRNA / MAPK6/MAPK4 signaling / Regulation of PTEN stability and activity / KEAP1-NFE2L2 pathway / Separation of Sister Chromatids / Antigen processing: Ubiquitination & Proteasome degradation / ABC-family proteins mediated transport / Ub-specific processing proteases / : / proteasome core complex / Neutrophil degranulation / immune system process / myofibril / NF-kappaB binding / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / negative regulation of inflammatory response to antigenic stimulus / response to organonitrogen compound / proteasome complex / proteolysis involved in protein catabolic process / sarcomere / ciliary basal body / proteasomal protein catabolic process / P-body / lipopolysaccharide binding / response to virus / response to organic cyclic compound / nuclear matrix / positive regulation of NF-kappaB transcription factor activity / peptidase activity / ubiquitin-dependent protein catabolic process / postsynapse / proteasome-mediated ubiquitin-dependent protein catabolic process / endopeptidase activity / response to oxidative stress / ribosome / centrosome / synapse / ubiquitin protein ligase binding / proteolysis / RNA binding / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Proteasome subunit alpha 1 / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / Proteasome beta subunit, C-terminal / Proteasome beta subunits C terminal / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 ...Proteasome subunit alpha 1 / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / Proteasome beta subunit, C-terminal / Proteasome beta subunits C terminal / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / 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 alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal / 4-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Proteasome subunit alpha type-2 / Proteasome subunit alpha type-1 / Proteasome subunit beta type-1 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-4 / Proteasome subunit beta type-6 / Proteasome subunit beta type-5 / Proteasome subunit alpha type-5 / Proteasome subunit beta type-4 / Proteasome subunit beta type-3 ...Proteasome subunit alpha type-2 / Proteasome subunit alpha type-1 / Proteasome subunit beta type-1 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-4 / Proteasome subunit beta type-6 / Proteasome subunit beta type-5 / Proteasome subunit alpha type-5 / Proteasome subunit beta type-4 / Proteasome subunit beta type-3 / Proteasome subunit beta type-2 / Proteasome subunit alpha type-7 / Proteasome subunit alpha type-6 / Proteasome subunit beta type-7
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsDeshmukh, F.K. / Polkinghorn, C.R. / Elad, N. / Sharon, M.
Funding support Israel, 2items
OrganizationGrant numberCountry
European Research Council (ERC)636752 Israel
Israel Science Foundation300/17 Israel
CitationJournal: ACS Cent Sci / Year: 2020
Title: Comparative Structural Analysis of 20S Proteasome Ortholog Protein Complexes by Native Mass Spectrometry.
Authors: Shay Vimer / Gili Ben-Nissan / David Morgenstern / Fanindra Kumar-Deshmukh / Caley Polkinghorn / Royston S Quintyn / Yury V Vasil'ev / Joseph S Beckman / Nadav Elad / Vicki H Wysocki / Michal Sharon /
Abstract: Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental ...Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multilevel experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast () and mammals (rat - , rabbit - and human - HEK293 cells), the proteasome increased both in size and stability. Native MS structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution, three-dimensional structures, highly resembled that of the human complex. Using cryoelectron microscopy single-particle analysis, we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.
History
DepositionJan 2, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 13, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Proteasome subunit alpha type-6
B: Proteasome subunit alpha type-2
C: Proteasome subunit alpha type-4
D: Proteasome subunit alpha type-7
E: Proteasome subunit alpha type-5
F: Proteasome subunit alpha type-1
G: Proteasome subunit alpha type-3
H: Proteasome subunit beta type-6
I: Proteasome subunit beta type-7
J: Proteasome subunit beta type-3
K: Proteasome subunit beta type-2
L: Proteasome subunit beta type-5
M: Proteasome subunit beta type-1
N: Proteasome subunit beta type-4
P: Proteasome subunit alpha type-2
T: Proteasome subunit alpha type-1
X: Proteasome subunit beta type-3
Y: Proteasome subunit beta type-2
Z: Proteasome subunit beta type-5
a: Proteasome subunit beta type-1
b: Proteasome subunit beta type-4
W: Proteasome subunit beta type-7
R: Proteasome subunit alpha type-7
O: Proteasome subunit alpha type-6
U: Proteasome subunit alpha type-3
S: Proteasome subunit alpha type-5
Q: Proteasome subunit alpha type-4
V: Proteasome subunit beta type-6


Theoretical massNumber of molelcules
Total (without water)762,56828
Polymers762,56828
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: scanning transmission electron microscopy, The structure of the mammalian 20S proteasome is known.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area93690 Å2
ΔGint-369 kcal/mol
Surface area236470 Å2

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Components

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Proteasome subunit alpha type- ... , 7 types, 14 molecules AOBPCQDRESFTGU

#1: Protein Proteasome subunit alpha type-6 / / Macropain iota chain / Multicatalytic endopeptidase complex iota chain / Proteasome iota chain


Mass: 27432.459 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P60901, proteasome endopeptidase complex
#2: Protein Proteasome subunit alpha type-2 / / Macropain subunit C3 / Multicatalytic endopeptidase complex subunit C3 / Proteasome component C3


Mass: 25955.549 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P17220, proteasome endopeptidase complex
#3: Protein Proteasome subunit alpha type-4 / / Macropain subunit C9 / Multicatalytic endopeptidase complex subunit C9 / Proteasome component C9 / ...Macropain subunit C9 / Multicatalytic endopeptidase complex subunit C9 / Proteasome component C9 / Proteasome subunit L


Mass: 29539.830 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P21670, proteasome endopeptidase complex
#4: Protein Proteasome subunit alpha type-7 / / Proteasome subunit RC6-1


Mass: 28369.439 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P48004, proteasome endopeptidase complex
#5: Protein Proteasome subunit alpha type-5 / / Macropain zeta chain / Multicatalytic endopeptidase complex zeta chain / Proteasome zeta chain


Mass: 26416.850 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P34064, proteasome endopeptidase complex
#6: Protein Proteasome subunit alpha type-1 / / Macropain subunit C2 / Multicatalytic endopeptidase complex subunit C2 / Proteasome component C2 / ...Macropain subunit C2 / Multicatalytic endopeptidase complex subunit C2 / Proteasome component C2 / Proteasome nu chain


Mass: 29557.541 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P18420, proteasome endopeptidase complex
#7: Protein Proteasome subunit alpha type-3 / / Macropain subunit C8 / Multicatalytic endopeptidase complex subunit C8 / Proteasome component C8 / ...Macropain subunit C8 / Multicatalytic endopeptidase complex subunit C8 / Proteasome component C8 / Proteasome subunit K


Mass: 28456.275 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P18422, proteasome endopeptidase complex

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Proteasome subunit beta type- ... , 7 types, 14 molecules HVIWJXKYLZMaNb

#8: Protein Proteasome subunit beta type-6 / / Macropain delta chain / Multicatalytic endopeptidase complex delta chain / Proteasome chain 5 / ...Macropain delta chain / Multicatalytic endopeptidase complex delta chain / Proteasome chain 5 / Proteasome delta chain / Proteasome subunit Y


Mass: 25309.576 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P28073, proteasome endopeptidase complex
#9: Protein Proteasome subunit beta type-7 / / Macropain chain Z / Multicatalytic endopeptidase complex chain Z / Proteasome subunit Z


Mass: 29963.461 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: Q9JHW0, proteasome endopeptidase complex
#10: Protein Proteasome subunit beta type-3 / PSMB3 / Proteasome chain 13 / Proteasome component C10-II / Proteasome theta chain


Mass: 22988.895 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P40112, proteasome endopeptidase complex
#11: Protein Proteasome subunit beta type-2 / PSMB2 / Macropain subunit C7-I / Multicatalytic endopeptidase complex subunit C7-I / Proteasome component C7-I


Mass: 22941.381 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P40307, proteasome endopeptidase complex
#12: Protein Proteasome subunit beta type-5 / PSMB5 / Macropain epsilon chain / Multicatalytic endopeptidase complex epsilon chain / Proteasome chain 6 / ...Macropain epsilon chain / Multicatalytic endopeptidase complex epsilon chain / Proteasome chain 6 / Proteasome epsilon chain / Proteasome subunit X


Mass: 28615.408 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P28075, proteasome endopeptidase complex
#13: Protein Proteasome subunit beta type-1 / PSMB1 / Macropain subunit C5 / Multicatalytic endopeptidase complex subunit C5 / Proteasome component C5 / ...Macropain subunit C5 / Multicatalytic endopeptidase complex subunit C5 / Proteasome component C5 / Proteasome gamma chain


Mass: 26511.301 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P18421, proteasome endopeptidase complex
#14: Protein Proteasome subunit beta type-4 / PSMB4 / Macropain beta chain / Multicatalytic endopeptidase complex beta chain / Proteasome beta chain / ...Macropain beta chain / Multicatalytic endopeptidase complex beta chain / Proteasome beta chain / Proteasome chain 3 / RN3


Mass: 29226.248 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Rattus norvegicus (Norway rat)
References: UniProt: P34067, proteasome endopeptidase complex

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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: Rat liver 20S proteasome / Type: COMPLEX
Details: Endogenous 20S proteasome purified from the rat livers by anion-exchange chromatography
Entity ID: all / Source: NATURAL
Source (natural)Organism: Rattus norvegicus (Norway rat)
Buffer solutionpH: 7.4
Buffer componentConc.: 160 mM / Name: Potassium phosphate / Formula: K3PO4
SpecimenConc.: 14 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Purified endogenous rat liver 20S proteasome
Specimen supportDetails: Grids were glow discharged 30 minutes before applying the sample.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 3 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Preliminary grid screening was performed on Talos Arctica
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: DIFFRACTION / Nominal magnification: 105000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 37 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2234
Details: Images were collected in movie-mode at 45 frames per second
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2EPU2image acquisition
4CTFFIND4CTF correction
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 284161
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 245800 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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