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Open data
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Basic information
Entry | Database: PDB / ID: 8cxb | ||||||||||||
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Title | Human PA28-20S (PA28-4a3b) | ||||||||||||
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![]() | HYDROLASE / proteasome / 20S / PA28 | ||||||||||||
Function / homology | ![]() proteasome activator complex / antigen processing and presentation of exogenous antigen / purine ribonucleoside triphosphate binding / regulation of endopeptidase activity / Regulation of ornithine decarboxylase (ODC) / proteasome core complex / Cross-presentation of soluble exogenous antigens (endosomes) / Somitogenesis / immune system process / myofibril ...proteasome activator complex / antigen processing and presentation of exogenous antigen / purine ribonucleoside triphosphate binding / regulation of endopeptidase activity / Regulation of ornithine decarboxylase (ODC) / proteasome core complex / Cross-presentation of soluble exogenous antigens (endosomes) / Somitogenesis / immune system process / myofibril / regulation of G1/S transition of mitotic cell cycle / NF-kappaB binding / endopeptidase activator activity / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / regulation of proteasomal protein catabolic process / negative regulation of inflammatory response to antigenic stimulus / : / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / sarcomere / proteasome complex / Regulation of activated PAK-2p34 by proteasome mediated degradation / ciliary basal body / proteolysis involved in protein catabolic process / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / Asymmetric localization of PCP proteins / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / Ubiquitin-dependent degradation of Cyclin D / AUF1 (hnRNP D0) binds and destabilizes mRNA / TNFR2 non-canonical NF-kB pathway / Vpu mediated degradation of CD4 / Assembly of the pre-replicative complex / Degradation of DVL / CDK-mediated phosphorylation and removal of Cdc6 / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Dectin-1 mediated noncanonical NF-kB signaling / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / lipopolysaccharide binding / SCF(Skp2)-mediated degradation of p27/p21 / Hh mutants are degraded by ERAD / Degradation of AXIN / Degradation of GLI1 by the proteasome / Activation of NF-kappaB in B cells / Hedgehog ligand biogenesis / Negative regulation of NOTCH4 signaling / Defective CFTR causes cystic fibrosis / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / G2/M Checkpoints / Vif-mediated degradation of APOBEC3G / Autodegradation of the E3 ubiquitin ligase COP1 / Hedgehog 'on' state / Regulation of RUNX3 expression and activity / P-body / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / Orc1 removal from chromatin / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / MAPK6/MAPK4 signaling / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / response to virus / Degradation of beta-catenin by the destruction complex / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / ABC-family proteins mediated transport / response to organic cyclic compound / CLEC7A (Dectin-1) signaling / Regulation of expression of SLITs and ROBOs / FCERI mediated NF-kB activation / nuclear matrix / Regulation of PTEN stability and activity / Interleukin-1 signaling / Regulation of RAS by GAPs / Separation of Sister Chromatids / Regulation of RUNX2 expression and activity / The role of GTSE1 in G2/M progression after G2 checkpoint / UCH proteinases / KEAP1-NFE2L2 pathway / Antigen processing: Ubiquitination & Proteasome degradation / regulation of inflammatory response / Downstream TCR signaling / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Neddylation / positive regulation of NF-kappaB transcription factor activity / peptidase activity / ER-Phagosome pathway / endopeptidase activity / proteasome-mediated ubiquitin-dependent protein catabolic process / secretory granule lumen / response to oxidative stress / ficolin-1-rich granule lumen / postsynapse / nuclear body / ribosome / Ub-specific processing proteases / cadherin binding / intracellular membrane-bounded organelle / centrosome Similarity search - Function | ||||||||||||
Biological species | ![]() | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||||||||
![]() | Zhao, J. / Makhija, S. / Huang, B. / Cheng, Y. | ||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Structural insights into the human PA28-20S proteasome enabled by efficient tagging and purification of endogenous proteins. Authors: Jianhua Zhao / Suraj Makhija / Chenyu Zhou / Hanxiao Zhang / YongQiang Wang / Monita Muralidharan / Bo Huang / Yifan Cheng / ![]() Abstract: The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically ...The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ-20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources. | ||||||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 1.3 MB | Display | ![]() |
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PDB format | ![]() | 1 MB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 24276MC ![]() 7nanC ![]() 7naoC ![]() 7napC ![]() 7naqC ![]() 8cvrC ![]() 8cvsC ![]() 8cvtC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Proteasome activator complex subunit ... , 2 types, 7 molecules cegdfhi
#1: Protein | Mass: 27439.633 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) ![]() #2: Protein | Mass: 28768.141 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) ![]() |
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-Proteasome subunit alpha type- ... , 7 types, 14 molecules AOBPCQDRESFTGU
#3: Protein | Mass: 25927.535 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P25787, proteasome endopeptidase complex #4: Protein | Mass: 29525.842 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P25789, proteasome endopeptidase complex #5: Protein | Mass: 27929.891 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: O14818, proteasome endopeptidase complex #6: Protein | Mass: 26435.977 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P28066, proteasome endopeptidase complex #7: Protein | Mass: 29595.627 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P25786, proteasome endopeptidase complex #8: Protein | Mass: 28469.252 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P25788, proteasome endopeptidase complex #9: Protein | Mass: 27432.459 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P60900, proteasome endopeptidase complex |
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-Proteasome subunit beta type- ... , 7 types, 14 molecules HVIWJXKYLZMaNb
#10: Protein | Mass: 30000.418 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: Q99436, proteasome endopeptidase complex #11: Protein | Mass: 22972.896 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P49720, proteasome endopeptidase complex #12: Protein | Mass: 22864.277 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P49721, proteasome endopeptidase complex #13: Protein | Mass: 28510.248 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P28074, proteasome endopeptidase complex #14: Protein | Mass: 26522.396 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P20618, proteasome endopeptidase complex #15: Protein | Mass: 29231.178 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P28070, proteasome endopeptidase complex #16: Protein | Mass: 25377.652 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P28072, proteasome endopeptidase complex |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: 20S-PA28(4a3b) / Type: COMPLEX / Entity ID: all / Source: NATURAL |
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Source (natural) | Organism: ![]() |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: FEI POLARA 300 |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1300 nm |
Image recording | Electron dose: 43 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
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Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135937 / Symmetry type: POINT |