+Open data
-Basic information
Entry | Database: PDB / ID: 6r70 | |||||||||
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Title | Endogeneous native human 20S proteasome | |||||||||
Components |
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Keywords | HYDROLASE / native / human / 20S proteasome / proteasome / cryo-EM / microfluidic / CryoWriter / protein isolation / endogeneous | |||||||||
Function / homology | Function and homology information 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 / NF-kappaB binding / proteasome endopeptidase complex ...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 / 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 / Regulation of activated PAK-2p34 by proteasome mediated degradation / ciliary basal body / 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 / Assembly of the pre-replicative complex / Vpu mediated degradation of CD4 / Degradation of DVL / proteasomal protein catabolic process / P-body / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Dectin-1 mediated noncanonical NF-kB signaling / Hh mutants are degraded by ERAD / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Degradation of AXIN / Defective CFTR causes cystic fibrosis / Degradation of GLI1 by the proteasome / lipopolysaccharide binding / Activation of NF-kappaB in B cells / Hedgehog ligand biogenesis / Negative regulation of NOTCH4 signaling / G2/M Checkpoints / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Autodegradation of the E3 ubiquitin ligase COP1 / Vif-mediated degradation of APOBEC3G / Hedgehog 'on' state / Regulation of RUNX3 expression and activity / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / MAPK6/MAPK4 signaling / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / response to virus / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / ABC-family proteins mediated transport / Degradation of beta-catenin by the destruction complex / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / response to organic cyclic compound / CDK-mediated phosphorylation and removal of Cdc6 / CLEC7A (Dectin-1) signaling / SCF(Skp2)-mediated degradation of p27/p21 / nuclear matrix / Regulation of expression of SLITs and ROBOs / FCERI mediated NF-kB activation / Regulation of PTEN stability and activity / Interleukin-1 signaling / Orc1 removal from chromatin / Regulation of RAS by GAPs / Separation of Sister Chromatids / Regulation of RUNX2 expression and activity / UCH proteinases / The role of GTSE1 in G2/M progression after G2 checkpoint / KEAP1-NFE2L2 pathway / Antigen processing: Ubiquitination & Proteasome degradation / Downstream TCR signaling / Neddylation / RUNX1 regulates transcription of genes involved in differentiation of HSCs / positive regulation of NF-kappaB transcription factor activity / peptidase activity / ER-Phagosome pathway / regulation of inflammatory response / postsynapse / proteasome-mediated ubiquitin-dependent protein catabolic process / secretory granule lumen / endopeptidase activity / response to oxidative stress / ficolin-1-rich granule lumen / nuclear body / ribosome / Ub-specific processing proteases / cadherin binding / intracellular membrane-bounded organelle / centrosome / synapse / ubiquitin protein ligase binding / Neutrophil degranulation / mitochondrion / proteolysis Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
Authors | Schmidli, C. / Albiez, S. / Rima, L. / Righetto, R. / Mohammed, I. / Oliva, P. / Kovacik, L. / Stahlberg, H. / Braun, T. | |||||||||
Funding support | Switzerland, 2items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2019 Title: Microfluidic protein isolation and sample preparation for high-resolution cryo-EM. Authors: Claudio Schmidli / Stefan Albiez / Luca Rima / Ricardo Righetto / Inayatulla Mohammed / Paolo Oliva / Lubomir Kovacik / Henning Stahlberg / Thomas Braun / Abstract: High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often ...High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6r70.cif.gz | 1 MB | Display | PDBx/mmCIF format |
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PDB format | pdb6r70.ent.gz | 886 KB | Display | PDB format |
PDBx/mmJSON format | 6r70.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/r7/6r70 ftp://data.pdbj.org/pub/pdb/validation_reports/r7/6r70 | HTTPS FTP |
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-Related structure data
Related structure data | 4738MC 4628C 6r7mC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | |
EM raw data | EMPIAR-10251 (Title: CryoWriter: 3.5 Å structure of human 20S proteasome with bound Fabs from microfluidic protein isolation, and 1.9 Å TMV structure Data size: 136.5 Data #1: Unaligned multi-frame micrographs of human 20S proteasome and TMV [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Proteasome subunit beta type- ... , 7 types, 14 molecules MaHVIWJXKYLZNb
#1: Protein | Mass: 24081.402 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P28070, proteasome endopeptidase complex #9: Protein | Mass: 23745.256 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: Q99436, proteasome endopeptidase complex #10: Protein | Mass: 22841.701 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P49720, proteasome endopeptidase complex #11: Protein | Mass: 22365.721 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P49721, proteasome endopeptidase complex #12: Protein | Mass: 22199.072 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P28074, proteasome endopeptidase complex #13: Protein | Mass: 23578.986 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P20618, proteasome endopeptidase complex #14: Protein | Mass: 21753.643 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P28072, proteasome endopeptidase complex |
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-Proteasome subunit alpha type- ... , 7 types, 14 molecules AOBPCQDRESFTGU
#2: Protein | Mass: 25525.068 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P25787, proteasome endopeptidase complex #3: Protein | Mass: 27859.896 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P25789, proteasome endopeptidase complex #4: Protein | Mass: 26623.291 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: O14818, proteasome endopeptidase complex #5: Protein | Mass: 25406.785 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P28066, proteasome endopeptidase complex #6: Protein | Mass: 26807.551 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P25786, proteasome endopeptidase complex #7: Protein | Mass: 26813.619 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P25788, proteasome endopeptidase complex #8: Protein | Mass: 27186.174 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P60900, 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 |
-Sample preparation
Component | Name: Native human 20S proteasome with bound Fabs to the two alpha-4 subunits Type: COMPLEX Details: Fab fragment generated by proteolytic cleavage of monoclonal IgG1 antibody. Entity ID: all / Source: NATURAL | |||||||||||||||||||||||||
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Molecular weight | Units: MEGADALTONS / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: Homo sapiens (human) | |||||||||||||||||||||||||
Buffer solution | pH: 7.4 / Details: PBS was purchased from Sigma # D8537, Switzerland. | |||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: 20S proteasome was purified using Fabs and eluted in a buffer containing 1mg/ml tobacco mosaic virus (TMV). The sample contains also unbound Fabs. | |||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE Details: CryoWriter. (see https://doi.org/10.1016/j.jsb.2016.11.002 for more information) |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 6 sec. / Electron dose: 72 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 523 |
Image scans | Width: 7676 / Height: 7420 / Movie frames/image: 30 / Used frames/image: 0-30 |
-Processing
Software | Name: PHENIX / Version: (1.14_3260: phenix.real_space_refine) / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 523 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16015 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5LE5 |