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- EMDB-2830: Electron cryotomography, subtomogram averaging and classification... -

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Basic information

Entry
Database: EMDB / ID: EMD-2830
TitleElectron cryotomography, subtomogram averaging and classification of 26S proteasomes in situ in intact hippocampal neurons
Map data26S Proteasome from mammal hippocampus neurons in ground state (GS)
Sample
  • Sample: 26S Proteasome from mammal hippocampus neurons in ground state (GS)
  • Protein or peptide: 26S Proteasome
KeywordsProteasome / 26S / 26S Proteasome / Ubiquitin / Ubiquitin-Proteasome Pathway / UPP / Ubiquitin-Proteasome System / UPS / Protease
Biological speciesRattus norvegicus (Norway rat)
Methodsubtomogram averaging / cryo EM / Resolution: 27.0 Å
AuthorsAsano S / Fukuda Y / Beck F / Aufderheide A / Foerster F / Danev R / Baumeister W
CitationJournal: Science / Year: 2015
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
DepositionDec 1, 2014-
Header (metadata) releaseJan 21, 2015-
Map releaseJan 21, 2015-
UpdateMar 16, 2016-
Current statusMar 16, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0277
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0277
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2830.tif

Downloads & links

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Map

FileDownload / File: emd_2830.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation26S Proteasome from mammal hippocampus neurons in ground state (GS)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.21 Å/pix.
x 128 pix.
= 538.88 Å		4.21 Å/pix.
x 128 pix.
= 538.88 Å		4.21 Å/pix.
x 128 pix.
= 538.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.21 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0277 / Movie #1: 0.0277
Minimum - Maximum-0.10450987 - 0.20428406
Average (Standard dev.)0.00071729 (±0.01649381)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 538.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.214.214.21
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z538.880538.880538.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1050.2040.001

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Supplemental data

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Sample components

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Entire : 26S Proteasome from mammal hippocampus neurons in ground state (GS)

EntireName: 26S Proteasome from mammal hippocampus neurons in ground state (GS)
Components
  • Sample: 26S Proteasome from mammal hippocampus neurons in ground state (GS)
  • Protein or peptide: 26S Proteasome

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Supramolecule #1000: 26S Proteasome from mammal hippocampus neurons in ground state (GS)

SupramoleculeName: 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

MacromoleculeName: 26S Proteasome / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Rattus norvegicus (Norway rat) / Strain: Wistar / synonym: Rat / Tissue: Brain / Location in cell: Cytoplasm

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

GridDetails: Quantifoil Au 200 mesh grids R1/4
VitrificationCryogen 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

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: Gatan
DetailsVolta Phase Plate
DateOct 24, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 110 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus min: 1.0 µm / Nominal magnification: 33000
Sample stageSpecimen 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 reconstructionApplied 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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