Lacey formvar carbon coated copper grids

For all peptides, fibrils were prepared from samples with monomer close to 5 $\mathsf{\mu }$ M total concentration, which were incubated at 37 ${}^{\circ }$ C in PEGylated plates (Corning 3881) in a plate reader and collected after reaching the plateau in ThT fluorescence. Specimens for cryo-TEM were prepared in an automatic plunge freezer system (Leica EM GP). The climate chamber temperature was kept at 21 ${}^{\circ }$ C, and relative humidity was ≥90% to minimise loss of solution during sample preparation. The specimens were prepared by placing 4 $\mathsf{\mu }$ L solution on glow discharged lacey formvar carbon coated copper grids (Ted Pella) and blotted with filter paper before being plunged into liquid ethane at −183 ${}^{\circ }$ C. This leads to vitrified specimens, avoiding component segmentation and rearrangement, and the formation of water crystals, thereby preserving original microstructures. The vitrified specimens were stored under liquid nitrogen until measured. A Fischione Model 2550 cryo transfer tomography holder was used to transfer the specimen into the electron microscope, JEM 2200FS, equipped with an in-column energy filter (Omega filter), which allows zero-loss imaging. The acceleration voltage was 200 kV and zero-loss images were recorded digitally with a TVIPS F416 camera using SerialEM under low dose conditions with a 10 eV energy selecting slit in place.

Cryo-TEM samples were prepared as follows. Lyophilized and desalted α-synuclein was resuspended in SUV dispersion. The lipid concentration was 20 mM in samples at L/P 200, 150, 100, 50, and 15 mM in the sample at L/P 25. The protein concentration was varied accordingly to reach the desired L/P ratio.
Specimens for cryo-TEM were prepared in an automatic plunge freezer system (Leica EM GP). The climate chamber temperature was kept at 21°C, and relative humidity was ≥90% to minimise loss of solution during sample preparation. The specimens were prepared by placing 4 μl solution on glow discharged lacey formvar carbon-coated copper grids (Ted Pella) and blotted with filter paper before being plunged into liquid ethane at −180°C. This leads to vitrified specimens, avoiding component segmentation and rearrangement, and the formation of water crystals, thereby preserving original microstructures. The vitrified specimens were stored under liquid nitrogen until measured. A Fischione Model 2550 cryo transfer tomography holder was used to transfer the specimen into the electron microscope, JEM 2200FS, equipped with an in-column energy filter (Omega filter), which allows zero-loss imaging. The acceleration voltage was 200 kV and zero-loss images were recorded digitally with a TVIPS F416 camera using SerialEM under low dose conditions with a 10 eV energy selecting slit in place.