Figure 1: Structural characterization of the two α-syn polymorphs.

(a) Time courses of α-syn (100 μM) assembly in buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl), red data points, and B (5 mM Tris-HCl, pH 7.5), blue data points, at 37 °C, monitored by measurement of scattered light at 440 nm. (b) Time courses of depolymerization at 4 °C of α-syn (100 μM monomer concentration) assemblies obtained in buffer A (high salt, red curve) and B (low salt, blue curve) assessed by quantifying α-syn within the pellet and supernatant fractions by SDS–PAGE, as described in the Methods. Data are mean±s.d. (n=4). (c,d) Negatively stained TEM of α-syn fibrils (c) and ribbons (d). The arrowheads point to twists; scale bars, 200 nm. (e,f), Proteinase K degradation patterns of α-syn (100 μM monomer concentration) fibrils (e) and ribbons (f), monitored over time on Coomassie stained SDS–PAGE (15%). Time (min) and molecular weight markers (kDa) are shown on the top and left of each gel, respectively. (g,h), X-ray diffraction pattern of partially aligned α-syn fibrils (g) and ribbons (h). α-syn fibrils X-ray scattering pattern is typical of amyloids showing a sharp anisotropic reflection at 4.7 Å along the meridian and another anisotropic reflection at 10 Å along the equator while that of α-syn ribbons resembles a powder/crystalline diffraction pattern with sharp meridional and equatorial reflections at 4.75 and 11 Å as well as long- and short-range reflections. (i) Radial averaging of the X-ray scattering patterns of α-syn fibrils (red curve) and ribbons (blue curve). (j) The conformational FILA antibody distinguishes equal amounts (0.4 μg) of α-syn fibrils (1) from α-syn ribbons (2) spotted on nitrocellulose membranes, whereas pan-α-syn antibodies (ASY1) do not. (k) Backbone Ψ angles as predicted by TALOS for α-syn fibrils (red) and ribbons (blue) based on sequential assignment. Suggested ß-sheet are indicated by arrows, with the lighter colours used for extensions if the glycines are assumed to be part of the ß-sheet or when uncertain TALOS β-sheet predictions (marked by crosses) are included into ß-sheets. Some residues in α-syn fibril show slight peak doubling. This is not considered here, as the resulting backbone angles are very similar.