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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 / response to desipramine / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process ...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 / response to desipramine / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / regulation of synaptic vesicle recycling / mitochondrial membrane organization / negative regulation of chaperone-mediated autophagy / 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 / dopamine biosynthetic process / response to iron(II) ion / positive regulation of neurotransmitter secretion / SNARE complex assembly / regulation of norepinephrine uptake / regulation of locomotion / negative regulation of dopamine metabolic process / positive regulation of inositol phosphate biosynthetic process / regulation of macrophage activation / transporter regulator activity / negative regulation of microtubule polymerization / synaptic vesicle transport / synaptic vesicle priming / dopamine uptake involved in synaptic transmission / protein kinase inhibitor activity / mitochondrial ATP synthesis coupled electron transport / regulation of dopamine secretion / dynein complex binding / positive regulation of receptor recycling / negative regulation of thrombin-activated receptor signaling pathway / cuprous ion binding / nuclear outer membrane / response to magnesium ion / positive regulation of endocytosis / positive regulation of exocytosis / synaptic vesicle exocytosis / kinesin binding / synaptic vesicle endocytosis / enzyme inhibitor activity / cysteine-type endopeptidase inhibitor activity / negative regulation of serotonin uptake / response to type II interferon / regulation of presynapse assembly / alpha-tubulin binding / beta-tubulin binding / phospholipase binding / behavioral response to cocaine / supramolecular fiber organization / phospholipid metabolic process / cellular response to fibroblast growth factor stimulus / inclusion body / axon terminus / Hsp70 protein binding / cellular response to epinephrine stimulus / response to interleukin-1 / regulation of microtubule cytoskeleton organization / cellular response to copper ion / positive regulation of release of sequestered calcium ion into cytosol / fatty acid binding / adult locomotory behavior / SNARE binding / excitatory postsynaptic potential / protein tetramerization / phosphoprotein binding / fatty acid metabolic process / microglial cell activation / ferrous iron binding / regulation of long-term neuronal synaptic plasticity / synapse organization / protein destabilization / PKR-mediated signaling / phospholipid binding / receptor internalization / tau protein binding / long-term synaptic potentiation / terminal bouton / positive regulation of inflammatory response / synaptic vesicle membrane / actin cytoskeleton / actin binding / growth cone / cellular response to oxidative stress / neuron apoptotic process / cell cortex / histone binding / response to lipopolysaccharide / microtubule binding / molecular adaptor activity / chemical synaptic transmission / amyloid fibril formation / mitochondrial outer membrane
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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