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- EMDB-10410: In situ subtomogram average of Chlamydomonas Cdc48 (C6 symmetry) -

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Basic information

Entry
Database: EMDB / ID: EMD-10410
TitleIn situ subtomogram average of Chlamydomonas Cdc48 (C6 symmetry)
Map dataIn situ subtomogram average of the Chlamydomonas Cdc48 complex (C6 symmetry)
Sample
  • Complex: Cdc48 inside native Chlamydomonas cells
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 32.8 Å
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
  • Surface view with section colored by density value
  • Surface level: 0.41
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.41
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10410.png

Downloads & links

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Map

FileDownload / File: emd_10410.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ subtomogram average of the Chlamydomonas Cdc48 complex (C6 symmetry)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.42 Å/pix.
x 96 pix.
= 328.32 Å		3.42 Å/pix.
x 96 pix.
= 328.32 Å		3.42 Å/pix.
x 96 pix.
= 328.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.42 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.41 / Movie #1: 0.41
Minimum - Maximum-0.14345692 - 0.7451323
Average (Standard dev.)0.0080864495 (±0.06989844)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 328.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.423.423.42
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z328.320328.320328.320
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-0.1430.7450.008

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Supplemental data

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Additional map: Raw subtomogram average before post-processing (C6 symmetry)

Fileemd_10410_additional.map
AnnotationRaw subtomogram average before post-processing (C6 symmetry)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cdc48 inside native Chlamydomonas cells

EntireName: Cdc48 inside native Chlamydomonas cells
Components
  • Complex: Cdc48 inside native Chlamydomonas cells

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Supramolecule #1: Cdc48 inside native Chlamydomonas cells

SupramoleculeName: Cdc48 inside native Chlamydomonas cells / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Location in cell: Cytoplasm
Molecular weightTheoretical: 540 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
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 the back for 10 seconds before plunging.

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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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 1.5 sec. / Average electron dose: 1.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 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 reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 32.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 789
ExtractionNumber tomograms: 76 / Number images used: 2075 / Reference model: Cdc48 structure filtered to 40A / Method: Template Matching / Software - Name: PyTom
CTF correctionSoftware - Name: IMOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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