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- EMDB-28753: Cryo-electron tomography of S42Y a-synuclein preformed fibrils -

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Entry
Database: EMDB / ID: EMD-28753
TitleCryo-electron tomography of S42Y a-synuclein preformed fibrils
Map dataCryo-electron tomography of S42Y a-synuclein preformed fibrils
Sample
  • Organelle or cellular component: S42Y a-synuclein preformed fibrils
Keywordsa-synuclein / preformed fibrils / PROTEIN FIBRIL
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodelectron tomography / cryo EM
AuthorsJiang J / Boparai N / Dai W / Kim YS
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)1R01-NS100919 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)1R01-NS101461 United States
CitationJournal: Acta Neuropathol / Year: 2023
Title: Transcriptional mutagenesis of α-synuclein caused by DNA oxidation in Parkinson's disease pathogenesis.
Authors: Sambuddha Basu / Minkyung Song / Levi Adams / Inhye Jeong / Goun Je / Subhrangshu Guhathakurta / Jennifer Jiang / Nikpreet Boparai / Wei Dai / Fernando Cardozo-Pelaez / Suren A Tatulian / ...Authors: Sambuddha Basu / Minkyung Song / Levi Adams / Inhye Jeong / Goun Je / Subhrangshu Guhathakurta / Jennifer Jiang / Nikpreet Boparai / Wei Dai / Fernando Cardozo-Pelaez / Suren A Tatulian / Kyu Young Han / Jordan Elliott / Jean Baum / Pamela J McLean / Dennis W Dickson / Yoon-Seong Kim /
Abstract: Oxidative stress plays an essential role in the development of Parkinson's disease (PD). 8-oxo-7,8-dihydroguanine (8-oxodG, oxidized guanine) is the most abundant oxidative stress-mediated DNA lesion. ...Oxidative stress plays an essential role in the development of Parkinson's disease (PD). 8-oxo-7,8-dihydroguanine (8-oxodG, oxidized guanine) is the most abundant oxidative stress-mediated DNA lesion. However, its contributing role in underlying PD pathogenesis remains unknown. In this study, we hypothesized that 8-oxodG can generate novel α-synuclein (α-SYN) mutants with altered pathologic aggregation through a phenomenon called transcriptional mutagenesis (TM). We observed a significantly higher accumulation of 8-oxodG in the midbrain genomic DNA from PD patients compared to age-matched controls, both globally and region specifically to α-SYN. In-silico analysis predicted that forty-three amino acid positions can contribute to TM-derived α-SYN mutation. Here, we report a significantly higher load of TM-derived α-SYN mutants from the midbrain of PD patients compared to controls using a sensitive PCR-based technique. We found a novel Serine42Tyrosine (S42Y) α-SYN as the most frequently detected TM mutant, which incidentally had the highest predicted aggregation score amongst all TM variants. Immunohistochemistry of midbrain sections from PD patients using a newly characterized antibody for S42Y identified S42Y-laden Lewy bodies (LB). We further demonstrated that the S42Y TM variant significantly accelerates WT α-SYN aggregation by cell and recombinant protein-based assays. Cryo-electron tomography revealed that S42Y exhibits considerable conformational heterogeneity compared to WT fibrils. Moreover, S42Y exhibited higher neurotoxicity compared to WT α-SYN as shown in mouse primary cortical cultures and AAV-mediated overexpression in the substantia nigra of C57BL/6 J mice. To our knowledge, this is the first report describing the possible contribution of TM-generated mutations of α-SYN to LB formation and PD pathogenesis.
History
DepositionNov 1, 2022-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28753.png

Downloads & links

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Map

FileDownload / File: emd_28753.map.gz / Format: CCP4 / Size: 238.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron tomography of S42Y a-synuclein preformed fibrils
Voxel sizeX=Y=Z: 8.536 Å
Density
Minimum - Maximum-72.762969999999996 - 52.964269999999999
Average (Standard dev.)0.0194919 (±1.588628)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-4-9-8
Dimensions92189276
Spacing89292176
CellA: 7614.1123 Å / B: 7861.6562 Å / C: 648.736 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : S42Y a-synuclein preformed fibrils

EntireName: S42Y a-synuclein preformed fibrils
Components
  • Organelle or cellular component: S42Y a-synuclein preformed fibrils

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Supramolecule #1: S42Y a-synuclein preformed fibrils

SupramoleculeName: S42Y a-synuclein preformed fibrils / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)

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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
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293.15 K / Instrument: LEICA EM GP / Details: Blot time: 3.5 seconds.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 3.5 µm / Nominal magnification: 63000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 41 / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: EMAN2 / Number images used: 41

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