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- EMDB-4191: In situ cryo-electron tomogram from Rat neuron with C9ORF72 Poly-... -

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Basic information

Entry
Database: EMDB / ID: EMD-4191
TitleIn situ cryo-electron tomogram from Rat neuron with C9ORF72 Poly-GA aggregates
Map dataTomogram of Rat neuron cell
Sample
  • Cell: in situ tomogram from Rat neuron
Biological speciesRattus norvegicus (Norway rat)
Methodelectron tomography / cryo EM
AuthorsGuo Q / Lehmer C / Martinez-Sanchez A / Rudack T / Beck F / Hartmann H / Hipp MS / Hartl FU / Edbauer D / Baumeister W / Fernandez-Busnadiego F
CitationJournal: Cell / Year: 2018
Title: In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment.
Authors: Qiang Guo / Carina Lehmer / Antonio Martínez-Sánchez / Till Rudack / Florian Beck / Hannelore Hartmann / Manuela Pérez-Berlanga / Frédéric Frottin / Mark S Hipp / F Ulrich Hartl / ...Authors: Qiang Guo / Carina Lehmer / Antonio Martínez-Sánchez / Till Rudack / Florian Beck / Hannelore Hartmann / Manuela Pérez-Berlanga / Frédéric Frottin / Mark S Hipp / F Ulrich Hartl / Dieter Edbauer / Wolfgang Baumeister / Rubén Fernández-Busnadiego /
Abstract: Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo- ...Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.
History
DepositionDec 5, 2017-
Header (metadata) releaseJan 17, 2018-
Map releaseFeb 7, 2018-
UpdateFeb 21, 2018-
Current statusFeb 21, 2018Processing site: PDBe / Status: Released

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Structure visualization

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

emd_4191.png

Downloads & links

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Map

FileDownload / File: emd_4191.map.gz / Format: CCP4 / Size: 232.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram of Rat neuron cell
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
13.68 Å/pix.
x 71 pix.
= 971.28 Å		13.68 Å/pix.
x 926 pix.
= 12667.681 Å		13.68 Å/pix.
x 926 pix.
= 12667.681 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: 13.68 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-21541.451000000000931 - 6443.688500000000204
Average (Standard dev.)0.25738844 (±464.516700000000014)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0011
Dimensions92692671
Spacing92692671
CellA: 12667.681 Å / B: 12667.681 Å / C: 971.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z13.68000107991413.68000107991413.68
M x/y/z92692671
origin x/y/z0.0000.0000.000
length x/y/z12667.68112667.681971.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS0011
NC/NR/NS92692671
D min/max/mean-21541.4516443.6880.257

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Supplemental data

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Sample components

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Entire : in situ tomogram from Rat neuron

EntireName: in situ tomogram from Rat neuron
Components
  • Cell: in situ tomogram from Rat neuron

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Supramolecule #1: in situ tomogram from Rat neuron

SupramoleculeName: in situ tomogram from Rat neuron / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1-#32
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
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: The grid was coated with C prior to use
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsFIB milled rat primary neurons
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.5 nA / Focused ion beam - Dose rate: 1 / Focused ion beam - Duration: 100 sec. / Focused ion beam - Temperature: 90 K / Focused ion beam - Initial thickness: 1500 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: 11nA rough milling. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Scios. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER ...Focused ion beam - Details: 11nA rough milling. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Scios. 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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Average exposure time: 2.0 sec. / Average electron dose: 1.8 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: 7.0 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 reconstructionResolution method: OTHER / Software - Name: eTomo / Number images used: 59

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