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- EMDB-10409: In situ cryo-electron tomogram from Chlamydomonas reinhardtii of ... -

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Entry
Database: EMDB / ID: EMD-10409
TitleIn situ cryo-electron tomogram from Chlamydomonas reinhardtii of a proteasome cluster at the endoplasmic reticulum
Map dataIn situ cryo-electron tomogram from Chlamydomonas reinhardtii, showing a cluster of proteasomes at the ER membrane.
Sample
  • Cell: Whole Chlamydomonas cells
Biological speciesChlamydomonas reinhardtii (plant)
Methodelectron tomography / cryo EM
AuthorsAlbert S / Schaffer M / Baumeister W / Engel BD
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2020
Title: Direct visualization of degradation microcompartments at the ER membrane.
Authors: Sahradha Albert / Wojciech Wietrzynski / Chia-Wei Lee / Miroslava Schaffer / Florian Beck / Jan M Schuller / Patrice A Salomé / Jürgen M Plitzko / Wolfgang Baumeister / Benjamin D Engel /
Abstract: To promote the biochemical reactions of life, cells can compartmentalize molecular interaction partners together within separated non-membrane-bound regions. It is unknown whether this strategy is ...To promote the biochemical reactions of life, cells can compartmentalize molecular interaction partners together within separated non-membrane-bound regions. It is unknown whether this strategy is used to facilitate protein degradation at specific locations within the cell. Leveraging in situ cryo-electron tomography to image the native molecular landscape of the unicellular alga , we discovered that the cytosolic protein degradation machinery is concentrated within ∼200-nm foci that contact specialized patches of endoplasmic reticulum (ER) membrane away from the ER-Golgi interface. These non-membrane-bound microcompartments exclude ribosomes and consist of a core of densely clustered 26S proteasomes surrounded by a loose cloud of Cdc48. Active proteasomes in the microcompartments directly engage with putative substrate at the ER membrane, a function canonically assigned to Cdc48. Live-cell fluorescence microscopy revealed that the proteasome clusters are dynamic, with frequent assembly and fusion events. We propose that the microcompartments perform ER-associated degradation, colocalizing the degradation machinery at specific ER hot spots to enable efficient protein quality control.
#1: Journal: To Be Published
Title: Direct visualization of degradation microcompartments at the ER membrane
Authors: Albert S / Wietrzynski W / Lee CW / Schaffer M / Beck F / Schuller JM / Salome PA / Plitzko JM / Baumeister W / Engel BD
History
DepositionOct 27, 2019-
Header (metadata) releaseNov 13, 2019-
Map releaseNov 13, 2019-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Movie viewer
Supplemental images

emd_10409.png

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Map

FileDownload / File: emd_10409.map.gz / Format: CCP4 / Size: 762.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationIn situ cryo-electron tomogram from Chlamydomonas reinhardtii, showing a cluster of proteasomes at the ER membrane.
Voxel sizeX=Y=Z: 13.68 Å
Density
Minimum - Maximum-89 - 76
Average (Standard dev.)2.5810552 (±10.353283)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00232
Dimensions928928464
Spacing928928464
CellA: 12695.04 Å / B: 12695.04 Å / C: 6347.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z13.6813.6813.68
M x/y/z928928464
origin x/y/z0.0000.0000.000
length x/y/z12695.04012695.0406347.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00232
NC/NR/NS928928464
D min/max/mean-89.00076.0002.581

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Supplemental data

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Sample components

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Entire : Whole Chlamydomonas cells

EntireName: Whole Chlamydomonas cells
Components
  • Cell: Whole Chlamydomonas cells

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Supramolecule #1: Whole Chlamydomonas cells

SupramoleculeName: Whole Chlamydomonas cells / type: cell / ID: 1 / Parent: 0
Details: Grown suspended in TAP media, with normal atmosphere aeration and constant light
Source (natural)Organism: Chlamydomonas reinhardtii (plant)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7 / Details: TAP media
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV / Details: blotted from back for 10 seconds before plunging.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Duration: 2000 sec. / Focused ion beam - Temperature: 95 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 150 nm
Focused ion beam - Details: See https://bio-protocol.org/e1575 for detailed procedure.. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Quanta FIB. This is not in a list of ...Focused ion beam - Details: See https://bio-protocol.org/e1575 for detailed procedure.. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Quanta FIB. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
DetailsBidirectional tilt scheme, separated at 0 degrees
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 1.5 sec. / Average electron dose: 100.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 5.6 µm / Calibrated defocus min: 4.6 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 60
CTF correctionSoftware - Name: IMOD

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