BiRD - Birkbeck Research Data

    EM images of alpha-synuclein amyloid fibres incubated with chaperones

    Cite as: MONISTROL, Jim and Saibil, Helen (2022): EM images of alpha-synuclein amyloid fibres incubated with chaperones. Birkbeck College, University of London. doi: https://doi.org/10.18743/DATA.00191

    Description

    The dataset contains a file describing the images and 5 EM images acquired on a F20 microscope.

    Collection Method

    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-/Ų.

    Data Objects

    Offline / Analogue Data Records

    There are no offline / analogue datasets associated with this record

    External Data Records

    There are no external datasets associated with this record

    Digital Data Downloads

    To download and items from this dataset, you must agree to abide by the licence attached to the individual items. If you make use of any item you download, you must also cite it in any publication or outputs of your own.

    If you have any questions or would like additional information, please contact us at researchdata@bbk.ac.uk.

    Full Archive

    Metadata

    Dataset Title:

    EM images of alpha-synuclein amyloid fibres incubated with chaperones

    Creators:

    MONISTROL, Jim and Saibil, Helen

    School/Department:

    Birkbeck Schools and Research Centres > School of Science > Biological Sciences

    Data collection method:

    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-/Ų.

    Statement on legal, ethical, and access issues:

    Not applicable

    Export / Share Citation

    Cite as: MONISTROL, Jim and Saibil, Helen (2022): EM images of alpha-synuclein amyloid fibres incubated with chaperones. Birkbeck College, University of London. doi: https://doi.org/10.18743/DATA.00191

    Impact & Reach

    Downloads
    Activity Overview
    28Downloads
    74Hits

    Additional statistics for this dataset are available via IRStats2.