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- EMDB-2991: Electron cryo-tomography of polyQ seeded mutant huntingtin aggreg... -

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Entry
Database: EMDB / ID: EMD-2991
TitleElectron cryo-tomography of polyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP
Map dataReconstruction of polyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP
Sample
  • Sample: PolyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP
  • Organelle or cellular component: mutant huntingtin aggregates
KeywordsHuntington's disease / seeding / cerebrospinal fluid / mutant huntingtin aggregates / cell lysate
Biological speciesRattus norvegicus (Norway rat)
Methodelectron tomography / cryo EM
AuthorsTan Z / Dai W / van Erp TGM / Overman J / Demuro A / Digman MA / Hatami A / Albay R / Sontag EM / Potkin KT ...Tan Z / Dai W / van Erp TGM / Overman J / Demuro A / Digman MA / Hatami A / Albay R / Sontag EM / Potkin KT / Ling S / Macciardi F / Bunney WE / Long JD / Paulsen JS / Ringman JM / Parker I / Glabe C / Thompson LM / Chiu W / Potkin SG
CitationJournal: Mol Psychiatry / Year: 2015
Title: Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin.
Authors: Z Tan / W Dai / T G M van Erp / J Overman / A Demuro / M A Digman / A Hatami / R Albay / E M Sontag / K T Potkin / S Ling / F Macciardi / W E Bunney / J D Long / J S Paulsen / J M Ringman / ...Authors: Z Tan / W Dai / T G M van Erp / J Overman / A Demuro / M A Digman / A Hatami / R Albay / E M Sontag / K T Potkin / S Ling / F Macciardi / W E Bunney / J D Long / J S Paulsen / J M Ringman / I Parker / C Glabe / L M Thompson / W Chiu / S G Potkin /
Abstract: Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. ...Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT.
History
DepositionApr 29, 2015-
Header (metadata) releaseJun 17, 2015-
Map releaseJul 15, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing site: PDBe / Status: Released

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Structure visualization

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  • Imaged by UCSF Chimera
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Supplemental images

EMD-2991.png

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Map

FileDownload / File: emd_2991.map.gz / Format: CCP4 / Size: 225.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of polyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP
Voxel sizeX=Y=Z: 28.6 Å
Density
Contour LevelBy EMDB: 60.0
Minimum - Maximum-1425.988159179999911 - 1030.745117189999974
Average (Standard dev.)19.078897479999998 (±48.196453089999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin68-190-10
Dimensions636780122
Spacing636780122
CellA: 22308.0 Å / B: 18189.6 Å / C: 3489.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z28.628.628.6
M x/y/z780636122
origin x/y/z0.0000.0000.000
length x/y/z22308.00018189.6003489.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-19068-10
NC/NR/NS780636122
D min/max/mean-1425.9881030.74519.079

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Supplemental data

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Sample components

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Entire : PolyQ seeded mutant huntingtin aggregates from PC12 cells inducib...

EntireName: PolyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP
Components
  • Sample: PolyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP
  • Organelle or cellular component: mutant huntingtin aggregates

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Supramolecule #1000: PolyQ seeded mutant huntingtin aggregates from PC12 cells inducib...

SupramoleculeName: PolyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP
type: sample / ID: 1000
Details: This sample is synthetic polyQ (KKQ40KK) seeded mutant huntingtin aggregates from PC12 cell lysates. These PC12 cells inducibly express mHTTex1-GFP.
Number unique components: 1

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Supramolecule #1: mutant huntingtin aggregates

SupramoleculeName: mutant huntingtin aggregates / type: organelle_or_cellular_component / ID: 1
Details: This sample is synthetic polyQ oligomer (KKQ40QQ) seeded mutant huntingtin aggregates from PC12 cell lysates. These cells inducibly express mHTTex1-GFP.
Oligomeric state: oligomers and fibrils / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Norway rat / Tissue: adrenal medulla / Cell: pheochromocytoma / Location in cell: cell lysate
Recombinant expressionOrganism: Rattus norvegicus (Norway rat) / Recombinant cell: PC12 cells / Recombinant plasmid: pIND

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

GridDetails: 200 mesh gold grid with thin carbon support, glow discharged for 20 second
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: LEICA EM GP / Method: Blot for 2 second before plunging

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 15000
Sample stageSpecimen holder: Gatan 626 70 degree / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 3 °
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateDec 12, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 40 / Average electron dose: 60 e/Å2 / Camera length: 1200

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Image processing

Final reconstructionAlgorithm: OTHER / Software - Name: IMOD / Details: Final map was reconstructed by back projection / Number images used: 40
Detailsalignment and reconstruction were done using IMOD

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