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- EMDB-32217: tomogram of a rat primary neuron harboring TDP-25 inclusion -

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Basic information

Entry
Database: EMDB / ID: EMD-32217
Titletomogram of a rat primary neuron harboring TDP-25 inclusion
Map data
Sample
  • Cell: rat primary neuron culture with TDP-25 inclusion
Biological speciesRattus norvegicus (Norway rat)
Methodelectron tomography / cryo EM
AuthorsGuo Q
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
CitationJournal: EMBO Rep / Year: 2022
Title: Gel-like inclusions of C-terminal fragments of TDP-43 sequester stalled proteasomes in neurons.
Authors: Henrick Riemenschneider / Qiang Guo / Jakob Bader / Frédéric Frottin / Daniel Farny / Gernot Kleinberger / Christian Haass / Matthias Mann / F Ulrich Hartl / Wolfgang Baumeister / Mark S ...Authors: Henrick Riemenschneider / Qiang Guo / Jakob Bader / Frédéric Frottin / Daniel Farny / Gernot Kleinberger / Christian Haass / Matthias Mann / F Ulrich Hartl / Wolfgang Baumeister / Mark S Hipp / Felix Meissner / Rubén Fernández-Busnadiego / Dieter Edbauer /
Abstract: Aggregation of the multifunctional RNA-binding protein TDP-43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both ...Aggregation of the multifunctional RNA-binding protein TDP-43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C-terminal fragments of ~25 kDa ("TDP-25") accumulate in cytoplasmic inclusions. Here, we analyze gain-of-function mechanisms of TDP-25 combining cryo-electron tomography, proteomics, and functional assays. In neurons, cytoplasmic TDP-25 inclusions are amorphous, and photobleaching experiments reveal gel-like biophysical properties that are less dynamic than nuclear TDP-43. Compared with full-length TDP-43, the TDP-25 interactome is depleted of low-complexity domain proteins. TDP-25 inclusions are enriched in 26S proteasomes adopting exclusively substrate-processing conformations, suggesting that inclusions sequester proteasomes, which are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP-25 impairs proteostasis, and this inhibitory function is enhanced by ALS-causing TDP-43 mutations. These findings support a patho-physiological relevance of proteasome dysfunction in ALS/FTD.
History
DepositionNov 15, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateJun 15, 2022-
Current statusJun 15, 2022Processing site: PDBj / 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_32217.png

Downloads & links

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Map

FileDownload / File: emd_32217.map.gz / Format: CCP4 / Size: 162.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 14.08 Å
Density
Minimum - Maximum-94538203000.0 - 27483766800.0
Average (Standard dev.)99011656.0 (±3683146500.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin004
Dimensions78078070
Spacing78078070
CellA: 10982.4 Å / B: 10982.4 Å / C: 985.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z14.0814.0814.08
M x/y/z78078070
origin x/y/z0.0000.0000.000
length x/y/z10982.40010982.400985.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS004
NC/NR/NS78078070
D min/max/mean-94538203136.00027483766784.00099011656.000

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Supplemental data

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Sample components

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Entire : rat primary neuron culture with TDP-25 inclusion

EntireName: rat primary neuron culture with TDP-25 inclusion
Components
  • Cell: rat primary neuron culture with TDP-25 inclusion

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Supramolecule #1: rat primary neuron culture with TDP-25 inclusion

SupramoleculeName: rat primary neuron culture with TDP-25 inclusion / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Rattus norvegicus (Norway rat)

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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
GridMaterial: GOLD
VitrificationCryogen name: ETHANE-PROPANE
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 1 nA / Focused ion beam - Duration: 1 sec. / Focused ion beam - Temperature: 100 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. 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 TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 2.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Number images used: 40

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