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- EMDB-6482: Cryo-electron microscopy of alpha Synuclein amyloid fibrils -

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Basic information

Entry
Database: EMDB / ID: EMD-6482
TitleCryo-electron microscopy of alpha Synuclein amyloid fibrils
Map dataExtruded 2D reconstruction of in vitro assembled alpha Synuclein amyoid fibrils
Sample
  • Sample: In vitro assembled, recombinant amyloid fibrils of full-length human alpha Synuclein
  • Protein or peptide: alpha Synuclein
Function / homology
Function and homology information


protein binding / negative regulation of mitochondrial electron transport, NADH to ubiquinone / : / neutral lipid metabolic process / regulation of acyl-CoA biosynthetic process / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber ...protein binding / negative regulation of mitochondrial electron transport, NADH to ubiquinone / : / neutral lipid metabolic process / regulation of acyl-CoA biosynthetic process / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / mitochondrial membrane organization / negative regulation of chaperone-mediated autophagy / regulation of synaptic vesicle recycling / regulation of reactive oxygen species biosynthetic process / negative regulation of platelet-derived growth factor receptor signaling pathway / positive regulation of protein localization to cell periphery / negative regulation of exocytosis / regulation of glutamate secretion / response to iron(II) ion / SNARE complex assembly / positive regulation of neurotransmitter secretion / dopamine biosynthetic process / regulation of norepinephrine uptake / transporter regulator activity / regulation of locomotion / synaptic vesicle priming / mitochondrial ATP synthesis coupled electron transport / regulation of macrophage activation / positive regulation of inositol phosphate biosynthetic process / negative regulation of microtubule polymerization / synaptic vesicle transport / positive regulation of receptor recycling / dopamine uptake involved in synaptic transmission / protein kinase inhibitor activity / dynein complex binding / regulation of dopamine secretion / negative regulation of thrombin-activated receptor signaling pathway / cuprous ion binding / positive regulation of endocytosis / positive regulation of exocytosis / response to magnesium ion / synaptic vesicle exocytosis / enzyme inhibitor activity / kinesin binding / synaptic vesicle endocytosis / regulation of presynapse assembly / response to type II interferon / cysteine-type endopeptidase inhibitor activity / negative regulation of serotonin uptake / alpha-tubulin binding / supramolecular fiber organization / inclusion body / phospholipid metabolic process / cellular response to copper ion / axon terminus / cellular response to epinephrine stimulus / Hsp70 protein binding / response to interleukin-1 / regulation of microtubule cytoskeleton organization / SNARE binding / positive regulation of release of sequestered calcium ion into cytosol / adult locomotory behavior / fatty acid binding / negative regulation of protein kinase activity / excitatory postsynaptic potential / fatty acid metabolic process / phosphoprotein binding / protein tetramerization / microglial cell activation / regulation of long-term neuronal synaptic plasticity / synapse organization / ferrous iron binding / protein destabilization / PKR-mediated signaling / phospholipid binding / receptor internalization / tau protein binding / long-term synaptic potentiation / synaptic vesicle membrane / positive regulation of inflammatory response / actin cytoskeleton / actin binding / growth cone / cell cortex / cellular response to oxidative stress / neuron apoptotic process / chemical synaptic transmission / molecular adaptor activity / negative regulation of neuron apoptotic process / response to lipopolysaccharide / histone binding / amyloid fibril formation / lysosome / oxidoreductase activity / postsynapse / transcription cis-regulatory region binding / positive regulation of apoptotic process / Amyloid fiber formation / copper ion binding / response to xenobiotic stimulus
Similarity search - Function
Alpha-synuclein / Synuclein / Alpha-synuclein / Synuclein
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 40.0 Å
AuthorsDearborn AD / Wall JS / Cheng N / Heymann JB / Kajava AV / Varkey J / Langen R / Steven AC
CitationJournal: J Biol Chem / Year: 2016
Title: α-Synuclein Amyloid Fibrils with Two Entwined, Asymmetrically Associated Protofibrils.
Authors: Altaira D Dearborn / Joseph S Wall / Naiqian Cheng / J Bernard Heymann / Andrey V Kajava / Jobin Varkey / Ralf Langen / Alasdair C Steven /
Abstract: Parkinson disease and other progressive neurodegenerative conditions are characterized by the intracerebral presence of Lewy bodies, containing amyloid fibrils of α-synuclein. We used cryo-electron ...Parkinson disease and other progressive neurodegenerative conditions are characterized by the intracerebral presence of Lewy bodies, containing amyloid fibrils of α-synuclein. We used cryo-electron microscopy and scanning transmission electron microscopy (STEM) to study in vitro-assembled fibrils. These fibrils are highly polymorphic. Focusing on twisting fibrils with an inter-crossover spacing of 77 nm, our reconstructions showed them to consist of paired protofibrils. STEM mass per length data gave one subunit per 0.47 nm axial rise per protofibril, consistent with a superpleated β-structure. The STEM images show two thread-like densities running along each of these fibrils, which we interpret as ladders of metal ions. These threads confirmed the two-protofibril architecture of the 77-nm twisting fibrils and allowed us to identify this morphotype in STEM micrographs. Some other, but not all, fibril morphotypes also exhibit dense threads, implying that they also present a putative metal binding site. We propose a molecular model for the protofibril and suggest that polymorphic variant fibrils have different numbers of protofibrils that are associated differently.
History
DepositionOct 14, 2015-
Header (metadata) releaseNov 25, 2015-
Map releaseDec 16, 2015-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.66
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.66
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.66
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6482.png

Downloads & links

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Map

FileDownload / File: emd_6482.map.gz / Format: CCP4 / Size: 14.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExtruded 2D reconstruction of in vitro assembled alpha Synuclein amyoid fibrils
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.54 Å/pix.
x 606 pix.
= 1539.24 Å		2.54 Å/pix.
x 80 pix.
= 203.2 Å		2.54 Å/pix.
x 80 pix.
= 203.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 2.54 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.66 / Movie #1: 3.66
Minimum - Maximum-3.53419471 - 8.32841015
Average (Standard dev.)0.33484867 (±2.09454417)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-400
Dimensions8080606
Spacing8080606
CellA: 203.2 Å / B: 203.2 Å / C: 1539.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z8080606
origin x/y/z0.0000.0000.000
length x/y/z203.200203.2001539.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-40-400
NC/NR/NS8080606
D min/max/mean-3.5348.3280.335

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Supplemental data

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Sample components

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Entire : In vitro assembled, recombinant amyloid fibrils of full-length hu...

EntireName: In vitro assembled, recombinant amyloid fibrils of full-length human alpha Synuclein
Components
  • Sample: In vitro assembled, recombinant amyloid fibrils of full-length human alpha Synuclein
  • Protein or peptide: alpha Synuclein

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Supramolecule #1000: In vitro assembled, recombinant amyloid fibrils of full-length hu...

SupramoleculeName: In vitro assembled, recombinant amyloid fibrils of full-length human alpha Synuclein
type: sample / ID: 1000 / Details: The sample was morphologically heterogeneous. / Oligomeric state: dimeric asymmetric unit / Number unique components: 2
Molecular weightMethod: Dark-field scanning transmission electron microscopy 59.1 MDa/micron compared to 61.5 MDa/micron theoretical weight

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Macromolecule #1: alpha Synuclein

MacromoleculeName: alpha Synuclein / type: protein_or_peptide / ID: 1 / Name.synonym: NACP, PARK1 / Oligomeric state: Amyloid / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Brain / Cell: Neuron / Location in cell: Lewy Body
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21*(DE3)pLysS / Recombinant plasmid: pRK172aS
SequenceUniProtKB: Alpha-synuclein
GO: magnesium ion binding, fatty acid binding, copper ion binding, calcium ion binding, protein binding
InterPro: Alpha-synuclein

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration4 mg/mL
BufferpH: 7.4 / Details: 10 mM HEPES, 100 mM NaCl, 0.1% NaN3
GridDetails: R1.2/1.3 400 mesh copper Quantifoil grid, glow-discharged in argon/oxygen
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90 K / Instrument: LEICA KF80 / Method: Manually blotted before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
DateJun 5, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 110 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51840 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsThe 2D reconstructions were made and aligned using bhelcross. The helical parameters were determined per fibril in real space and imposed on the average using bhelcross.
Final reconstructionApplied symmetry - Helical parameters - Δz: 4.7 Å
Applied symmetry - Helical parameters - Δ&Phi: 1.1 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: OTHER / Software - Name: Bsoft
Details: Extruded from a 2D reconstruction based upon helical parameters
CTF correctionDetails: phase-flipped micrograph

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