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- EMDB-3095: Electron negative stain tomography of alpha-synuclein amyloid fib... -

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Basic information

Entry
Database: EMDB / ID: EMD-3095
TitleElectron negative stain tomography of alpha-synuclein amyloid fibrils treated with the Hsc70/DNAJB1/Apg2 chaperone system
Map dataReconstruction of alpha-synuclein amyloid fibrils treated with the Hsc70/DNAJB1/Apg2 trichaperone system
Sample
  • Sample: alpha-synuclein fibrils plus chaperones Hsc70, DNAJB1 and Apg2
  • Protein or peptide: alpha-synuclein
  • Protein or peptide: Hsc70
  • Protein or peptide: DNAJB1
  • Protein or peptide: Apg2
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 / response to iron(II) ion / positive regulation of neurotransmitter secretion / 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 / positive regulation of inositol phosphate biosynthetic process / synaptic vesicle priming / regulation of norepinephrine uptake / transporter regulator activity / protein kinase inhibitor activity / dopamine uptake involved in synaptic transmission / synaptic vesicle transport / regulation of dopamine secretion / positive regulation of receptor recycling / positive regulation of exocytosis / mitochondrial ATP synthesis coupled electron transport / cuprous ion binding / nuclear outer membrane / dynein complex binding / response to magnesium ion / synaptic vesicle exocytosis / positive regulation of endocytosis / negative regulation of serotonin uptake / response to type II interferon / kinesin binding / cysteine-type endopeptidase inhibitor activity / regulation of presynapse assembly / synaptic vesicle endocytosis / alpha-tubulin binding / beta-tubulin binding / phospholipase binding / supramolecular fiber organization / phospholipid metabolic process / behavioral response to cocaine / cellular response to fibroblast growth factor stimulus / inclusion body / cellular response to epinephrine stimulus / Hsp70 protein binding / enzyme inhibitor activity / axon terminus / response to interleukin-1 / cellular response to copper ion / positive regulation of release of sequestered calcium ion into cytosol / regulation of microtubule cytoskeleton organization / SNARE binding / adult locomotory behavior / glutathione metabolic process / protein tetramerization / excitatory postsynaptic potential / protein sequestering activity / tubulin binding / phosphoprotein binding / microglial cell activation / ferrous iron binding / fatty acid metabolic process / PKR-mediated signaling / synapse organization / receptor internalization / regulation of long-term neuronal synaptic plasticity / phospholipid binding / protein destabilization / tau protein binding / enzyme activator activity / terminal bouton / positive regulation of inflammatory response / 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

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

EMD-3095.png

Downloads & links

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Map

FileDownload / File: emd_3095.map.gz / Format: CCP4 / Size: 2.8 GB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationReconstruction of alpha-synuclein amyloid fibrils treated with the Hsc70/DNAJB1/Apg2 trichaperone system
Voxel sizeX=Y=Z: 3.028 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)0.48000404 (±23.18089676)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0096
Dimensions46563492192
Spacing46563492192
CellA: 10573.776 Å / B: 14098.368 Å / C: 581.37604 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/z34924656192
origin x/y/z0.0000.0000.000
length x/y/z10573.77614098.368581.376
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS0096
NC/NR/NS34924656192
D min/max/mean-128.000127.0000.480

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Supplemental data

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Sample components

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Entire : alpha-synuclein fibrils plus chaperones Hsc70, DNAJB1 and Apg2

EntireName: alpha-synuclein fibrils plus chaperones Hsc70, DNAJB1 and Apg2
Components
  • Sample: alpha-synuclein fibrils plus chaperones Hsc70, DNAJB1 and Apg2
  • Protein or peptide: alpha-synuclein
  • Protein or peptide: Hsc70
  • Protein or peptide: DNAJB1
  • Protein or peptide: Apg2

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Supramolecule #1000: alpha-synuclein fibrils plus chaperones Hsc70, DNAJB1 and Apg2

SupramoleculeName: alpha-synuclein fibrils plus chaperones Hsc70, DNAJB1 and Apg2
type: sample / ID: 1000 / Number unique components: 4

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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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Macromolecule #2: Hsc70

MacromoleculeName: Hsc70 / type: protein_or_peptide / ID: 2 / Name.synonym: HSPA8 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 70 KDa / Theoretical: 70 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #3: DNAJB1

MacromoleculeName: DNAJB1 / type: protein_or_peptide / ID: 3 / Name.synonym: Hdj1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 40 KDa / Theoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #4: Apg2

MacromoleculeName: Apg2 / type: protein_or_peptide / ID: 4 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 95 KDa / Theoretical: 95 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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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