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- EMDB-3094: Electron negative stain tomography of alpha-synuclein amyloid fibrils -

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Basic information

Entry
Database: EMDB / ID: EMD-3094
TitleElectron negative stain tomography of alpha-synuclein amyloid fibrils
Map datanegative stain tomogram of alpha-synuclein fibrils
Sample
  • Sample: alpha-synuclein fibrils formed in vitro
  • Protein or peptide: alpha-synuclein
Keywordsalpha-synuclein amyloid fibrils / disaggregation activity
Function / homology
Function and homology information


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 ...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 / negative regulation of chaperone-mediated autophagy / regulation of reactive oxygen species biosynthetic process / positive regulation of protein localization to cell periphery / mitochondrial membrane organization / negative regulation of exocytosis / regulation of glutamate secretion / dopamine biosynthetic process / positive regulation of neurotransmitter secretion / response to iron(II) ion / regulation of macrophage activation / negative regulation of dopamine metabolic process / negative regulation of platelet-derived growth factor receptor signaling pathway / SNARE complex assembly / negative regulation of thrombin-activated receptor signaling pathway / Lewy body / regulation of locomotion / negative regulation of microtubule polymerization / synaptic vesicle priming / regulation of norepinephrine uptake / transporter regulator activity / protein kinase inhibitor activity / positive regulation of inositol phosphate biosynthetic process / synaptic vesicle transport / dopamine uptake involved in synaptic transmission / regulation of dopamine secretion / positive regulation of receptor recycling / cuprous ion binding / positive regulation of exocytosis / nuclear outer membrane / mitochondrial ATP synthesis coupled electron transport / dynein complex binding / synaptic vesicle exocytosis / response to magnesium ion / positive regulation of endocytosis / negative regulation of serotonin uptake / response to type II interferon / cysteine-type endopeptidase inhibitor activity / kinesin binding / regulation of presynapse assembly / synaptic vesicle endocytosis / alpha-tubulin binding / beta-tubulin binding / phospholipase binding / phospholipid metabolic process / supramolecular fiber organization / behavioral response to cocaine / cellular response to fibroblast growth factor stimulus / cellular response to epinephrine stimulus / inclusion body / Hsp70 protein binding / enzyme inhibitor activity / response to interleukin-1 / axon terminus / cellular response to copper ion / regulation of microtubule cytoskeleton organization / positive regulation of release of sequestered calcium ion into cytosol / SNARE binding / adult locomotory behavior / glutathione metabolic process / protein tetramerization / protein sequestering activity / tubulin binding / phosphoprotein binding / excitatory postsynaptic potential / microglial cell activation / ferrous iron binding / fatty acid metabolic process / phospholipid binding / PKR-mediated signaling / synapse organization / receptor internalization / regulation of long-term neuronal synaptic plasticity / protein destabilization / tau protein binding / enzyme activator activity / positive regulation of inflammatory response / terminal bouton / long-term synaptic potentiation / synaptic vesicle membrane / actin cytoskeleton / growth cone / actin binding / cellular response to oxidative stress / neuron apoptotic process / cell cortex / histone binding / response to lipopolysaccharide / microtubule binding / amyloid fibril formation / chemical synaptic transmission
Similarity search - Function
Synuclein / Alpha-synuclein / Synuclein
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodelectron tomography / negative staining
AuthorsGao X / Carroni M / Nussbaum-Krammer C / Mogk A / Nillegoda NB / Szlachcic A / Guilbride DL / Saibil HR / Mayer MP / Bukau B
CitationJournal: Mol Cell / Year: 2015
Title: Human Hsp70 Disaggregase Reverses Parkinson's-Linked α-Synuclein Amyloid Fibrils.
Authors: Xuechao Gao / Marta Carroni / Carmen Nussbaum-Krammer / Axel Mogk / Nadinath B Nillegoda / Anna Szlachcic / D Lys Guilbride / Helen R Saibil / Matthias P Mayer / Bernd Bukau /
Abstract: Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early ...Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson's disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases.
History
DepositionJul 13, 2015-
Header (metadata) releaseAug 12, 2015-
Map releaseSep 16, 2015-
UpdateSep 16, 2015-
Current statusSep 16, 2015Processing site: PDBe / Status: Released

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Structure visualization

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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
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Structure viewerEM map:
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Supplemental images

EMD-3094.png

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Map

FileDownload / File: emd_3094.map.gz / Format: CCP4 / Size: 2.7 GB / Type: IMAGE STORED AS SIGNED BYTE
Annotationnegative stain tomogram of alpha-synuclein fibrils
Voxel sizeX=Y=Z: 3.028 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)6.72140121 (±21.095314030000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0080
Dimensions46563492184
Spacing46563492184
CellA: 10573.776 Å / B: 14098.368 Å / C: 557.15204 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z3.0283.0283.028
M x/y/z34924656184
origin x/y/z0.0000.0000.000
length x/y/z10573.77614098.368557.152
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS0080
NC/NR/NS34924656184
D min/max/mean-128.000127.0006.721

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Supplemental data

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Sample components

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Entire : alpha-synuclein fibrils formed in vitro

EntireName: alpha-synuclein fibrils formed in vitro
Components
  • Sample: alpha-synuclein fibrils formed in vitro
  • Protein or peptide: alpha-synuclein

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Supramolecule #1000: alpha-synuclein fibrils formed in vitro

SupramoleculeName: alpha-synuclein fibrils formed in vitro / type: sample / ID: 1000 / Oligomeric state: amyloid fibers / Number unique components: 1

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Macromolecule #1: alpha-synuclein

MacromoleculeName: alpha-synuclein / type: protein_or_peptide / ID: 1 / Oligomeric state: amyloid fibrils / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 14 KDa / Theoretical: 14 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pTYB11
SequenceUniProtKB: Alpha-synuclein

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Details: 50 mM Hepes-KOH pH 7.5, 50 mM KCl, 5 mM MgCl2, 2 mM DTT, 2 mM ATP, 3 mM PEP, 20 ng/microL pyruvate kinase
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl formate for 2 minutes
GridDetails: 200 mesh copper finder grids with thin carbon support glow discharged in atmosphere.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
DateDec 7, 2014
Image recordingCategory: CCD / Film or detector model: OTHER / Average electron dose: 200 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 3 °
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Software - Name: IMOD / Number images used: 40
CTF correctionDetails: IMOD

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