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.