+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27018 | |||||||||
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Title | Human 19S-20S proteasome, state SD2 | |||||||||
Map data | ||||||||||
Sample |
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Function / homology | Function and homology information positive regulation of inclusion body assembly / Impaired BRCA2 translocation to the nucleus / Impaired BRCA2 binding to SEM1 (DSS1) / thyrotropin-releasing hormone receptor binding / modulation by host of viral transcription / Hydrolases; Acting on peptide bonds (peptidases); Omega peptidases / proteasome accessory complex / purine ribonucleoside triphosphate binding / integrator complex / meiosis I ...positive regulation of inclusion body assembly / Impaired BRCA2 translocation to the nucleus / Impaired BRCA2 binding to SEM1 (DSS1) / thyrotropin-releasing hormone receptor binding / modulation by host of viral transcription / Hydrolases; Acting on peptide bonds (peptidases); Omega peptidases / proteasome accessory complex / purine ribonucleoside triphosphate binding / integrator complex / meiosis I / positive regulation of proteasomal protein catabolic process / proteasome regulatory particle / cytosolic proteasome complex / proteasome regulatory particle, lid subcomplex / proteasome-activating activity / proteasome regulatory particle, base subcomplex / protein K63-linked deubiquitination / metal-dependent deubiquitinase activity / regulation of endopeptidase activity / negative regulation of programmed cell death / Defective homologous recombination repair (HRR) due to BRCA1 loss of function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function / Homologous DNA Pairing and Strand Exchange / Regulation of ornithine decarboxylase (ODC) / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / proteasome core complex / Resolution of D-loop Structures through Holliday Junction Intermediates / Cross-presentation of soluble exogenous antigens (endosomes) / Somitogenesis / K63-linked deubiquitinase activity / Impaired BRCA2 binding to RAD51 / immune system process / myofibril / proteasome binding / regulation of protein catabolic process / proteasome storage granule / Presynaptic phase of homologous DNA pairing and strand exchange / transcription factor binding / blastocyst development / general transcription initiation factor binding / NF-kappaB binding / endopeptidase activator activity / proteasome assembly / polyubiquitin modification-dependent protein binding / proteasome endopeptidase complex / positive regulation of RNA polymerase II transcription preinitiation complex assembly / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / mRNA export from nucleus / enzyme regulator activity / regulation of proteasomal protein catabolic process / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / ERAD pathway / inclusion body / negative regulation of inflammatory response to antigenic stimulus / : / sarcomere / proteasome complex / Regulation of activated PAK-2p34 by proteasome mediated degradation / ciliary basal body / proteolysis involved in protein catabolic process / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / 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 / stem cell differentiation / 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 Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) / human (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.0 Å | |||||||||
Authors | Zhao J | |||||||||
Funding support | 1 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2022 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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27018.map.gz | 16 MB | EMDB map data format | |
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Header (meta data) | emd-27018-v30.xml emd-27018.xml | 50.4 KB 50.4 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_27018_fsc.xml | 20.8 KB | Display | FSC data file |
Images | emd_27018.png | 98.4 KB | ||
Others | emd_27018_half_map_1.map.gz emd_27018_half_map_2.map.gz | 475.7 MB 475.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27018 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27018 | HTTPS FTP |
-Related structure data
Related structure data | 8cvtMC 7nanC 7naoC 7napC 7naqC 8cvrC 8cvsC 8cxbC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_27018.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.06 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_27018_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_27018_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Human 19S-20S proteasome
+Supramolecule #1: Human 19S-20S proteasome
+Macromolecule #1: 26S proteasome regulatory subunit 7
+Macromolecule #2: 26S proteasome regulatory subunit 4
+Macromolecule #3: 26S protease regulatory subunit 8
+Macromolecule #4: 26S proteasome regulatory subunit 6B
+Macromolecule #5: 26S protease regulatory subunit 10B
+Macromolecule #6: 26S proteasome regulatory subunit 6A
+Macromolecule #7: Proteasome subunit alpha type-6
+Macromolecule #8: Proteasome subunit alpha type-2
+Macromolecule #9: Proteasome subunit alpha type-4
+Macromolecule #10: Proteasome subunit alpha type-7
+Macromolecule #11: Proteasome subunit alpha type-5
+Macromolecule #12: Proteasome subunit alpha type-1
+Macromolecule #13: Proteasome subunit alpha type-3
+Macromolecule #14: Proteasome subunit beta type-6
+Macromolecule #15: Proteasome subunit beta type-7
+Macromolecule #16: Proteasome subunit beta type-3
+Macromolecule #17: Proteasome subunit beta type-2
+Macromolecule #18: Proteasome subunit beta type-5
+Macromolecule #19: Proteasome subunit beta type-1
+Macromolecule #20: Proteasome subunit beta type-4
+Macromolecule #21: 26S proteasome non-ATPase regulatory subunit 1
+Macromolecule #22: 26S proteasome non-ATPase regulatory subunit 3
+Macromolecule #23: 26S proteasome non-ATPase regulatory subunit 12
+Macromolecule #24: 26S proteasome non-ATPase regulatory subunit 11
+Macromolecule #25: 26S proteasome non-ATPase regulatory subunit 6
+Macromolecule #26: 26S proteasome non-ATPase regulatory subunit 7
+Macromolecule #27: 26S proteasome non-ATPase regulatory subunit 13
+Macromolecule #28: 26S proteasome non-ATPase regulatory subunit 4
+Macromolecule #29: 26S proteasome non-ATPase regulatory subunit 14
+Macromolecule #30: 26S proteasome non-ATPase regulatory subunit 8
+Macromolecule #31: 26S proteasome complex subunit SEM1
+Macromolecule #32: 26S proteasome non-ATPase regulatory subunit 2
+Macromolecule #33: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #34: N-[(benzyloxy)carbonyl]-L-leucyl-N-[(2S)-4-methyl-1-oxopentan-2-y...
+Macromolecule #35: ZINC ION
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 30.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |