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- EMDB-51460: Tomogram of aggregate in AgDD-sfGFP-expressing HEK293 cell 6 h po... -

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Basic information

Entry
Database: EMDB / ID: EMD-51460
TitleTomogram of aggregate in AgDD-sfGFP-expressing HEK293 cell 6 h post aggregation induction
Map dataTomogram of aggregate in AgDD-sfGFP-expressing HEK293 cell 6 h post aggregation induction
Sample
  • Cell: Aggregate in AgDD-sfGFP-expressing HEK293 cell 6 h post aggregation induction
KeywordsAgDD / Aggregate / PROTEIN BINDING
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsSchaefer T / Fernandez-Busnadiego R
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.
Authors: Kelsey L Hickey / Alexandra Panov / Enya Miguel Whelan / Tillman Schäfer / Arda Mizrak / Ron R Kopito / Wolfgang Baumeister / Rubén Fernández-Busnadiego / J Wade Harper /
Abstract: A hallmark of neurodegenerative diseases (NDs) is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To ...A hallmark of neurodegenerative diseases (NDs) is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.
History
DepositionSep 2, 2024-
Header (metadata) releaseOct 23, 2024-
Map releaseOct 23, 2024-
UpdateMay 7, 2025-
Current statusMay 7, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51460.png

Downloads & links

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Map

FileDownload / File: emd_51460.map.gz / Format: CCP4 / Size: 410.6 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationTomogram of aggregate in AgDD-sfGFP-expressing HEK293 cell 6 h post aggregation induction
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
20.88 Å/pix.
x 250 pix.
= 5220. Å		20.88 Å/pix.
x 928 pix.
= 19376.639 Å		20.88 Å/pix.
x 928 pix.
= 19376.639 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 20.88 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-10307.0 - 4209.0
Average (Standard dev.)310.073939999999993 (±216.944179999999989)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions928928250
Spacing928928250
CellA: 19376.639 Å / B: 19376.639 Å / C: 5220.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Aggregate in AgDD-sfGFP-expressing HEK293 cell 6 h post aggregati...

EntireName: Aggregate in AgDD-sfGFP-expressing HEK293 cell 6 h post aggregation induction
Components
  • Cell: Aggregate in AgDD-sfGFP-expressing HEK293 cell 6 h post aggregation induction

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Supramolecule #1: Aggregate in AgDD-sfGFP-expressing HEK293 cell 6 h post aggregati...

SupramoleculeName: Aggregate in AgDD-sfGFP-expressing HEK293 cell 6 h post aggregation induction
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
VitrificationCryogen name: ETHANE-PROPANE
Cryo protectant10 % glycerol
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.1 / Focused ion beam - Duration: 300 / Focused ion beam - Temperature: 77 K / Focused ion beam - Initial thickness: 250 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is FEI Quanta FIB. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 9.0 µm / Nominal defocus min: 9.0 µm
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber images used: 110
CTF correctionType: NONE

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