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Yorodumi- EMDB-2991: Electron cryo-tomography of polyQ seeded mutant huntingtin aggreg... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2991 | |||||||||
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Title | Electron cryo-tomography of polyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP | |||||||||
Map data | Reconstruction of polyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP | |||||||||
Sample |
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Keywords | Huntington's disease / seeding / cerebrospinal fluid / mutant huntingtin aggregates / cell lysate | |||||||||
Biological species | Rattus norvegicus (Norway rat) | |||||||||
Method | electron tomography / cryo EM | |||||||||
Authors | Tan 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 | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2991.map.gz | 212.8 MB | EMDB map data format | |
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Header (meta data) | emd-2991-v30.xml emd-2991.xml | 11.1 KB 11.1 KB | Display Display | EMDB header |
Images | EMD-2991.png | 461.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2991 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2991 | HTTPS FTP |
-Validation report
Summary document | emd_2991_validation.pdf.gz | 169.9 KB | Display | EMDB validaton report |
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Full document | emd_2991_full_validation.pdf.gz | 169.1 KB | Display | |
Data in XML | emd_2991_validation.xml.gz | 5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2991 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2991 | HTTPS FTP |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_2991.map.gz / Format: CCP4 / Size: 225.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of polyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 28.6 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : PolyQ seeded mutant huntingtin aggregates from PC12 cells inducib...
Entire | Name: PolyQ seeded mutant huntingtin aggregates from PC12 cells inducibly expressing mHTTex1-GFP |
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Components |
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-Supramolecule #1000: PolyQ seeded mutant huntingtin aggregates from PC12 cells inducib...
Supramolecule | Name: 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
Supramolecule | Name: 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 |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) / synonym: Norway rat / Tissue: adrenal medulla / Cell: pheochromocytoma / Location in cell: cell lysate |
Recombinant expression | Organism: Rattus norvegicus (Norway rat) / Recombinant cell: PC12 cells / Recombinant plasmid: pIND |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
Aggregation state | cell |
-Sample preparation
Grid | Details: 200 mesh gold grid with thin carbon support, glow discharged for 20 second |
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Instrument: LEICA EM GP / Method: Blot for 2 second before plunging |
-Electron microscopy
Microscope | JEOL 2100 |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification |
Date | Dec 12, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 40 / Average electron dose: 60 e/Å2 / Camera length: 1200 |
Electron beam | Acceleration voltage: 200 kV / Electron source: LAB6 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 15000 |
Sample stage | Specimen 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 ° |
-Image processing
Details | alignment and reconstruction were done using IMOD |
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Final reconstruction | Algorithm: OTHER / Software - Name: IMOD / Details: Final map was reconstructed by back projection / Number images used: 40 |