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- EMDB-2830: Electron cryotomography, subtomogram averaging and classification... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2830 | |||||||||
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Title | Electron cryotomography, subtomogram averaging and classification of 26S proteasomes in situ in intact hippocampal neurons | |||||||||
![]() | 26S Proteasome from mammal hippocampus neurons in ground state (GS) | |||||||||
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![]() | Proteasome / 26S / 26S Proteasome / Ubiquitin / Ubiquitin-Proteasome Pathway / UPP / Ubiquitin-Proteasome System / UPS / Protease | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 27.0 Å | |||||||||
![]() | Asano S / Fukuda Y / Beck F / Aufderheide A / Foerster F / Danev R / Baumeister W | |||||||||
![]() | ![]() Title: Proteasomes. A molecular census of 26S proteasomes in intact neurons. Authors: Shoh Asano / Yoshiyuki Fukuda / Florian Beck / Antje Aufderheide / Friedrich Förster / Radostin Danev / Wolfgang Baumeister / ![]() Abstract: The 26S proteasome is a key player in eukaryotic protein quality control and in the regulation of numerous cellular processes. Here, we describe quantitative in situ structural studies of this highly ...The 26S proteasome is a key player in eukaryotic protein quality control and in the regulation of numerous cellular processes. Here, we describe quantitative in situ structural studies of this highly dynamic molecular machine in intact hippocampal neurons. We used electron cryotomography with the Volta phase plate, which allowed high fidelity and nanometer precision localization of 26S proteasomes. We undertook a molecular census of single- and double-capped proteasomes and assessed the conformational states of individual complexes. Under the conditions of the experiment—that is, in the absence of proteotoxic stress—only 20% of the 26S proteasomes were engaged in substrate processing. The remainder was in the substrate-accepting ground state. These findings suggest that in the absence of stress, the capacity of the proteasome system is not fully used. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 7.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 8.3 KB 8.3 KB | Display Display | ![]() |
Images | ![]() | 1.2 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 26S Proteasome from mammal hippocampus neurons in ground state (GS) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.21 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : 26S Proteasome from mammal hippocampus neurons in ground state (GS)
Entire | Name: 26S Proteasome from mammal hippocampus neurons in ground state (GS) |
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Components |
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-Supramolecule #1000: 26S Proteasome from mammal hippocampus neurons in ground state (GS)
Supramolecule | Name: 26S Proteasome from mammal hippocampus neurons in ground state (GS) type: sample / ID: 1000 / Number unique components: 1 |
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-Macromolecule #1: 26S Proteasome
Macromolecule | Name: 26S Proteasome / type: protein_or_peptide / ID: 1 / Recombinant expression: No |
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Source (natural) | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | subtomogram averaging |
Aggregation state | particle |
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Sample preparation
Grid | Details: Quantifoil Au 200 mesh grids R1/4 |
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Vitrification | Cryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK III Method: 37 deg C, waiting time 5 sec, and 10 seconds blot time |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: Gatan |
Details | Volta Phase Plate |
Date | Oct 24, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 110 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus min: 1.0 µm / Nominal magnification: 33000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 27.0 Å / Resolution method: OTHER / Software - Name: etomo, TOM, PYTOM / Number subtomograms used: 1367 |
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