EM images of alpha-synuclein amyloid fibres incubated with chaperones

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Cryo-EM_3-chaperones.tif

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NS_3-chaperones_WT-DNAJ.tif

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http://eprints.org/relation/isVersionOf https://researchdata.bbk.ac.uk/id/document/1037 http://eprints.org/relation/isVolatileVersionOf https://researchdata.bbk.ac.uk/id/document/1037 http://eprints.org/relation/issmallThumbnailVersionOf https://researchdata.bbk.ac.uk/id/document/1037 archive 126 disk0/00/00/01/91 2022-04-07 13:00:57 2026-04-14 11:12:21 2022-04-07 13:00:57 data_collection show MONISTROL Jim Department of Biological Sciences, Birkbeck College 0000-0002-4634-818X Saibil Helen Department of Biological Sciences, Birkbeck College 0000-0002-2266-8891 EM images of alpha-synuclein amyloid fibres incubated with chaperones NS bcs public The dataset contains a file describing the images and 5 EM images acquired on a F20 microscope. 2022-04-04 Birkbeck College, University of London 10.18743/DATA.00191 yes ismb metadata_and_data_files yes Three independent repeats were performed for each condition. Cryo-EM sample preparation: alpha-synuclein (αSyn) amyloid fibres were sonicated for 30 minutes using a Branson 1800 ultrasonic cleaner to produce dispersed fragments of αSyn fibres. The suspension was diluted to 6 µM αSyn monomer concentration and mixed, when indicated, with 6 µM Hsc70, 3 µM DNAJB1 and 0.6 µM Apg2, and incubated for 1 hour at 30°C in disaggregation buffer (or HKMD buffer for the control without chaperones). 4 µL of this preparation were applied to negatively glow discharged holey carbon C-flat grids (CF-1.2/1.3-4C) (Protochips, USA), back blotted and plunge frozen in liquid ethane using a Leica EM GP2 (Leica Microsystems, Germany). Negative stain sample preparation: αSyn fibres (10 µM monomer) were mixed, when indicated, with Hsc70 (10 µM), Apg2 (1 µM), and WT or ΔJ-DNAJB1(5 µM monomer) for 1 hour at 30°C in the disaggregation buffer (or HKMD buffer for the control without chaperones). These samples were applied to glow discharged continuous carbon film-coated, 300 mesh copper grids (EMS) and blotted after 1 minute. The grid was then stained with 2% (w/v) uranyl formate and immediately blotted 3 times. Images were acquired using a Tecnai TF20 electron microscope (Thermo Fisher, USA) equipped with FEG source operating at 200 keV. Images were recorded with a DE-20 direct electron detector (Direct Electron, USA) at a magnification corresponding to 1.83 Å/pixel using a total dose of ~30 e-/Å². Not applicable