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- EMDB-13576: Alpha-synuclein amyloid fibres incubated with DNAJB1, Hsc70, Hsp1... -

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Entry
Database: EMDB / ID: EMD-13576
TitleAlpha-synuclein amyloid fibres incubated with DNAJB1, Hsc70, Hsp110 and ATP
Map dataAlpha-synuclein amyloid fibres incubated with Hsc70, DNAJB1, Apg2 and ATP show extended stretches of densely clustered chaperones.
Sample
  • Complex: Complex of alpha-synuclein amyloid fibres, DNAJB1, Hsc70 and Hsp110/Apg2 chaperones
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsMonistrol J / Saibil HR
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust206166/Z/17/Z United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
CitationJournal: EMBO J / Year: 2022
Title: Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase.
Authors: Joseph George Beton / Jim Monistrol / Anne Wentink / Erin C Johnston / Anthony John Roberts / Bernd Gerhard Bukau / Bart W Hoogenboom / Helen R Saibil /
Abstract: Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. ...Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure.
History
DepositionSep 10, 2021-
Header (metadata) releaseJun 29, 2022-
Map releaseJun 29, 2022-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_13576.map.gz / Format: CCP4 / Size: 1.3 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAlpha-synuclein amyloid fibres incubated with Hsc70, DNAJB1, Apg2 and ATP show extended stretches of densely clustered chaperones.
Voxel sizeX=Y=Z: 8.52 Å
Density
Minimum - Maximum-7577.2314 - 4679.8257
Average (Standard dev.)102.27678 (±112.444145)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin161-208140
Dimensions13511024255
Spacing10241351255
CellA: 8724.48 Å / B: 11510.5205 Å / C: 2172.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Complex of alpha-synuclein amyloid fibres, DNAJB1, Hsc70 and Hsp1...

EntireName: Complex of alpha-synuclein amyloid fibres, DNAJB1, Hsc70 and Hsp110/Apg2 chaperones
Components
  • Complex: Complex of alpha-synuclein amyloid fibres, DNAJB1, Hsc70 and Hsp110/Apg2 chaperones

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Supramolecule #1: Complex of alpha-synuclein amyloid fibres, DNAJB1, Hsc70 and Hsp1...

SupramoleculeName: Complex of alpha-synuclein amyloid fibres, DNAJB1, Hsc70 and Hsp110/Apg2 chaperones
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mmol/LHEPES
50.0 mmol/LKClpotassium chloride
5.0 mmol/LMgCl2magnesium chloride
2.0 mmol/LDTT1,4-Dithiothreitol
5.0 mmol/LATPAdenosine triphosphateadenosine 5'-triphosphate
6.0 mmol/LPEPphosphoenol-pyruvate
20.0 ng/uLpyruvate kinase
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 42000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.0 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 150.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD (ver. 4.9.0) / Number images used: 41

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