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- EMDB-11401: Tomogram of GFP-a-synuclein inclusion in primary mouse neuron exp... -

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Basic information

Entry
Database: EMDB / ID: EMD-11401
TitleTomogram of GFP-a-synuclein inclusion in primary mouse neuron expressing GFP-a-synuclein, seeded with PFFs
Map dataTomogram of aggregate in primary neuron expressing GFP-a-syn, seeded with PFFs
Sample
  • Cell: GFP-a-synuclein inclusion in primary mouse neuron expressing GFP-a-synuclein, seeded with PFFs
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsTrinkaus VA / Hartl FU / Fernandez-Busnadiego R
Funding support Germany, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)FP7 GA ERC-2012-SyG_318987-ToPAG Germany
German Research Foundation (DFG)EXC 2067/1- 390729940 Germany
CitationJournal: Nat Commun / Year: 2021
Title: In situ architecture of neuronal α-Synuclein inclusions.
Authors: Victoria A Trinkaus / Irene Riera-Tur / Antonio Martínez-Sánchez / Felix J B Bäuerlein / Qiang Guo / Thomas Arzberger / Wolfgang Baumeister / Irina Dudanova / Mark S Hipp / F Ulrich Hartl ...Authors: Victoria A Trinkaus / Irene Riera-Tur / Antonio Martínez-Sánchez / Felix J B Bäuerlein / Qiang Guo / Thomas Arzberger / Wolfgang Baumeister / Irina Dudanova / Mark S Hipp / F Ulrich Hartl / Rubén Fernández-Busnadiego /
Abstract: The molecular architecture of α-Synuclein (α-Syn) inclusions, pathognomonic of various neurodegenerative disorders, remains unclear. α-Syn inclusions were long thought to consist mainly of α-Syn ...The molecular architecture of α-Synuclein (α-Syn) inclusions, pathognomonic of various neurodegenerative disorders, remains unclear. α-Syn inclusions were long thought to consist mainly of α-Syn fibrils, but recent reports pointed to intracellular membranes as the major inclusion component. Here, we use cryo-electron tomography (cryo-ET) to image neuronal α-Syn inclusions in situ at molecular resolution. We show that inclusions seeded by α-Syn aggregates produced recombinantly or purified from patient brain consist of α-Syn fibrils crisscrossing a variety of cellular organelles. Using gold-labeled seeds, we find that aggregate seeding is predominantly mediated by small α-Syn fibrils, from which cytoplasmic fibrils grow unidirectionally. Detailed analysis of membrane interactions revealed that α-Syn fibrils do not contact membranes directly, and that α-Syn does not drive membrane clustering. Altogether, we conclusively demonstrate that neuronal α-Syn inclusions consist of α-Syn fibrils intermixed with membranous organelles, and illuminate the mechanism of aggregate seeding and cellular interaction.
History
DepositionJul 16, 2020-
Header (metadata) releaseJan 20, 2021-
Map releaseJan 20, 2021-
UpdateAug 4, 2021-
Current statusAug 4, 2021Processing 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_11401.png

Downloads & links

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Map

FileDownload / File: emd_11401.map.gz / Format: CCP4 / Size: 348.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationTomogram of aggregate in primary neuron expressing GFP-a-syn, seeded with PFFs
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
17.56 Å/pix.
x 212 pix.
= 3722.72 Å		17.56 Å/pix.
x 928 pix.
= 16295.68 Å		17.56 Å/pix.
x 928 pix.
= 16295.68 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: 17.56 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-512.0 - 267.0
Average (Standard dev.)2.2597933 (±12.686158)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin10600
Dimensions928928212
Spacing928928212
CellA: 16295.68 Å / B: 16295.68 Å / C: 3722.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z17.5617.5617.56
M x/y/z928928212
origin x/y/z0.0000.0000.000
length x/y/z16295.68016295.6803722.720
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS01060
NC/NR/NS928928212
D min/max/mean-512.000267.0002.260

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Supplemental data

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Sample components

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Entire : GFP-a-synuclein inclusion in primary mouse neuron expressing GFP-...

EntireName: GFP-a-synuclein inclusion in primary mouse neuron expressing GFP-a-synuclein, seeded with PFFs
Components
  • Cell: GFP-a-synuclein inclusion in primary mouse neuron expressing GFP-a-synuclein, seeded with PFFs

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Supramolecule #1: GFP-a-synuclein inclusion in primary mouse neuron expressing GFP-...

SupramoleculeName: GFP-a-synuclein inclusion in primary mouse neuron expressing GFP-a-synuclein, seeded with PFFs
type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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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: 7.4
GridModel: Quantifoil R2/4 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 80 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV
Cryo protectant10 % Glycerol
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.1 nA / Focused ion beam - Duration: 2400 sec. / Focused ion beam - Temperature: 77 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 150 nm
Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Quanta FIB. 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
TemperatureMin: 70.0 K / Max: 75.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 21-28 / Number grids imaged: 1 / Number real images: 55 / Average exposure time: 4.0 sec. / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 7.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 34000
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 reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.9.0) / Number images used: 55

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