Fastscan d ss probes

Mica was freshly cleaved before being treated to create a positive surface charge by adding poly-l-lysine (70–150 kDa) at 15 µg/mL for 10 s followed by drying with nitrogen. A sample volume of 90 μL of protein (WT αSyn, L38M, Y39A or S42A) was taken at the end point of a fibril growth assay (as described above) before being deposited at a concentration of 30 μM onto poly-l-lysine treated mica and allowed to incubate for 4 min. The mica surface was then rinsed with buffer (50 mM sodium phosphate buffer, 300 mM KCl, pH 7.5) via fluid exchange, maintaining the samples in a liquid environment. AFM observations were performed in liquid in tapping mode using a Dimension FastScan Bio with FastScan-D-SS probes (Bruker) in the same buffer. The force applied by the tip on the sample was minimized by maximizing the set point whilst maintaining tracking of the surface. Heights of single particles were measured automatically using routines written in MATLAB (https://github.com/George-R-Heath/Particle-Detect). Heights and lengths of fibrils were measured either automatically using MATLAB (https://github.com/George-R-Heath/Correlate-Filaments) or manually in ImageJ for densely packed overlapping fibrils.

The detailed procedure for the AFM image acquisition is described elsewhere³⁴ . Here is a brief outline of the procedure. P1, P2 and P3 particle images (Extended Data Fig. 3) were recorded under the solution conditions described above using a Cypher VRS AFM (Asylum Research, Oxford Instrument) at 4°C with amplitude-modulated AC mode at a scan rate of 1 Hz (commonly known as tapping mode) using FASTSCAN-D-SS probes (Bruker, CA). For RNA immobilization, 50 mM APS stock was freshly diluted 300-fold in deionized water right before use and then used to coat a freshly cleaved muscovite mica (Grade V1) (Ted Pella Redding, CA) and incubated for 30 min, followed by rinsing the mica surface with deionized water and drying gently with filtered nitrogen gas.