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- EMDB-10494: cryo-ET of cryo-FIB milled HCT116 cell in the nuclear region, fol... -

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Basic information

Entry
Database: EMDB / ID: EMD-10494
Titlecryo-ET of cryo-FIB milled HCT116 cell in the nuclear region, following 0.1M sucrose stimulation. Shown in Fig.1b of publication
Map data
Sample
  • Cell: nuclear region of HCT116 cell, following 0.2M sucrose stimulation
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsGuo Q / Yasuda S / Baumeister W / Fernandez-Busnadiego R / Saeki Y
Funding support Japan, Germany, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science18K19352 Japan
European CommissionFP7 GA ERC-2012-SyG_318987-ToPAG Germany
CitationJournal: Nature / Year: 2020
Title: Stress- and ubiquitylation-dependent phase separation of the proteasome.
Authors: Sayaka Yasuda / Hikaru Tsuchiya / Ai Kaiho / Qiang Guo / Ken Ikeuchi / Akinori Endo / Naoko Arai / Fumiaki Ohtake / Shigeo Murata / Toshifumi Inada / Wolfgang Baumeister / Rubén Fernández- ...Authors: Sayaka Yasuda / Hikaru Tsuchiya / Ai Kaiho / Qiang Guo / Ken Ikeuchi / Akinori Endo / Naoko Arai / Fumiaki Ohtake / Shigeo Murata / Toshifumi Inada / Wolfgang Baumeister / Rubén Fernández-Busnadiego / Keiji Tanaka / Yasushi Saeki /
Abstract: The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins. A number of ubiquitin-related molecules have recently been ...The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes, but it remains unclear whether the proteasome also participates in such regulation. Here we reveal that proteasome-containing nuclear foci form under acute hyperosmotic stress. These foci are transient structures that contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP)) and multiple proteasome-interacting proteins, which collectively constitute a proteolytic centre. The major substrates for degradation by these foci were ribosomal proteins that failed to properly assemble. Notably, the proteasome foci exhibited properties of liquid droplets. RAD23B, a substrate-shuttling factor for the proteasome, and ubiquitylated proteins were necessary for formation of proteasome foci. In mechanistic terms, a liquid-liquid phase separation was triggered by multivalent interactions of two ubiquitin-associated domains of RAD23B and ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our results suggest that ubiquitin-chain-dependent phase separation induces the formation of a nuclear proteolytic compartment that promotes proteasomal degradation.
History
DepositionNov 15, 2019-
Header (metadata) releaseJul 22, 2020-
Map releaseJul 22, 2020-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing 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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Supplemental images

emd_10494.png

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Map

FileDownload / File: emd_10494.map.gz / Format: CCP4 / Size: 1.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 13.68 Å
Density
Minimum - Maximum-18667.459 - 6336.0264
Average (Standard dev.)-0.0030212512 (±277.39514)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions926926375
Spacing926926375
CellA: 12667.681 Å / B: 12667.681 Å / C: 5130.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z13.68000107991413.68000107991413.68
M x/y/z926926375
origin x/y/z0.0000.0000.000
length x/y/z12667.68112667.6815130.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS926926375
D min/max/mean-18667.4596336.026-0.003

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Supplemental data

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Sample components

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Entire : nuclear region of HCT116 cell, following 0.2M sucrose stimulation

EntireName: nuclear region of HCT116 cell, following 0.2M sucrose stimulation
Components
  • Cell: nuclear region of HCT116 cell, following 0.2M sucrose stimulation

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Supramolecule #1: nuclear region of HCT116 cell, following 0.2M sucrose stimulation

SupramoleculeName: nuclear region of HCT116 cell, following 0.2M sucrose stimulation
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.01 nA / Focused ion beam - Duration: 1 sec. / Focused ion beam - Temperature: 93 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 200 nm
Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is Quanta 3D FEG, FEI. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 14620 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 7.0 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 reconstructionSoftware: (Name: RELION, IMOD) / Number images used: 60
CTF correctionSoftware:
Namedetails
RELIONFOR CORRECTION
CTFFINDFOR DETERMINE

Details: this is done by RELION

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