Nature Structural & Molecular Biology: doi: /nsmb.3194

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1 Supplementary Figure 1 Mass spectrometry and solution NMR data for -syn samples used in this study. (a) Matrix-assisted laser-desorption and ionization time-of-flight (MALDI-TOF) mass spectrum of uniformly- 13 C, 15 N- enriched -syn monomer (blue) and natural abundance monomer (red). The molecular mass of natural abundance -syn monomer was calculated to be kda, in agreement with the experimental mass spectrum. The mass of kda for the U- 13 C, 15 N-labeled sample corresponded to a ~98% incorporation of 13 C and 15 N during protein expression. (b) 15 N- 1 H HSQC spectrum of monomeric -syn prior to fibrillization. The chemical shifts agreed well with the published assignments (Eliezer, D., Kutluay, E., Bussell, R. & Browne, G. Conformational properties of α-synuclein in its free and lipid-associated states. J. Mol. Biol. 307, (2001).)

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3 Supplementary Figure 2 SSNMR spectroscopy data for -syn fibrils, exhibiting cross-peaks indicative of long-range restraints and intermolecular registry of the fibrils. (a) 2D 13 C- 13 C SSNMR { 1 H}- 13 C-{ 1 H- 1 H}- 13 C spectrum (Lange, A., Luca, S. & Baldus, M. Structural constraints from proton-mediated rare-spin correlation spectroscopy in rotating solids. J. Am. Chem. Soc. 124, (2002).) of dilute -syn fibrils (sample D from Table 1) showing unambiguous, long-range intramolecular correlations. Data were acquired at 750 MHz 1 H frequency, 12.5 khz MAS and 400 s 1 H- 1 H mixing time, with 12 days signal averaging. Red labels correspond to unambiguous long-range distances, including those from A78 to V48, from A69 to G93, from F94 to I88, and from V82 to A89. These intramolecular long-range restraints were inconsistent with structural models that exhibit a domain swap. (b-e) 2D 15 N, 13 C SSNMR transferred echo double resonance (TEDOR, Jaroniec, C.P., Filip, C. & Griffin, R.G. 3D TEDOR NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly 13 C, 15 N-labeled solids. J. Am. Chem. Soc. 124, (2002).) data showing intermolecular interactions. The data were collected for four samples: (b) 1,3-13 C glycerol, 15 N-labeled fibrils (sample B), signal averaged for 28 hr; (c) 50:50 [1,3-13 C]glycerol, natural abundance (n.a.) nitrogen: n.a. carbon, 15 N labeled (sample E), signal averaged for 81 hr; (d) [2-13 C]glycerol, 15 N labeled (sample C), signal averaged for 113 hr; and (e) 50: C glycerol, n.a. nitrogen: n.a. carbon, 15 N labeled (sample F), signal averaged for 124 hr. Data was collected with mixing times of (c, e) 14.4 ms at 500 MHz 1 H frequency, 11.1 khz MAS, and (b, d) 16.0 ms at 600 MHz 1 H frequency, 10.0 khz MAS. Cross-peaks present in both parts b and c, or d and e, demonstrate a parallel, in-register arrangement (Debelouchina, G.T., Platt, G.W., Bayro, M.J., Radford, S.E. & Griffin, R.G. Intermolecular alignment in 2 -microglobulin amyloid fibrils. J. Am. Chem. Soc. 132, (2010).) (f k) Spectra of sample B collected at 750 MHz 1 H frequency, 12.5 khz MAS (f-i) 2D 13 C- 13 C spectra using dipolar-assisted rotational resonance (DARR, Takegoshi, K., Nakamura, S. & Terao, T. 13 C- 1 H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem. Phys. Lett. 344, (2001).) mixing times of (f) 50 ms, signal averaged for 10.9 hr, (g) 100 ms, signal averaged for 5.7 hr, (h) 200 ms, signal averaged for 12.0 hr, and (i) 300 ms, signal averaged for 12.7 hr. Several long-range correlations (red) were observed between the F94 aromatic ring resonances (CD and CE) and I88, A90 and A91. (j-k) 2D planes from a 3D 15 N- 13 CO- 13 CX correlation spectrum signal averaged for 24 hr. Long-range correlations observed at this relatively short mixing time indicate internuclear distances <5 Å.

4 Supplementary Figure 3 Full-length structure of the -syn fibril, illustrating important features in expanded regions. (a) View along the fibril axis showing the highly ordered core and the disordered tails. (b) Side view showing the -sheet packing between each monomer as well as the disordered tails. (c-f) Core interactions illustrating NMR distance restraints with dotted lines. (g-l) Backbone traces for neighboring monomers drawn in blue, yellow, orange and red. (g, h) Side and top views of the salt bridge from E46 to K80 of the neighboring monomer. (i, j) Side and top views of the I88-A91-F94 pocket perpendicular to the fibril axis, exhibiting short intermolecular distances. (k, l) Side and top view of the Q79 side chain exhibiting a glutamine ladder, with intermolecular hydrogen bonds involving Nε2 and Oε1 moieties.

5 Supplementary Figure 4 SSNMR internal validation of structural restraints and dihedral angles for the -syn fibril structure. (a) The backbone trace is shown in grey, and black lines represent unambiguous distance restraints. Blue lines correspond to the shortest observed distance among the possible ambiguous assignments. The resulting structure is the lowest energy conformer consistent with all of the data. (b-c) Ramachandran probability maps of accepted dihedral angle regions (Lovell, S.C. et al. Structure validation by C geometry:, and C deviation. Proteins 50, (2003).) and plotted in Chimera (Pettersen, E.F. et al. UCSF Chimera a visualization system for exploratory research and analysis. J. Comput. Chem. 25, (2004).) for residues The only residue not in the accepted range is E57, which is part of an unstructured loop and lacks NMR restraints in the simulated annealing calculations. All glycine residues are within the accepted Ramachandran space.

6 Supplementary Figure 5 Cross-validation of the SSNMR structure of -syn fibrils with electron microscopy and X-ray fiber diffraction. (a) Chemical shift comparison of the fibril sample for solid-state NMR before and after washing with 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) buffer and sonication for electron microscopy. (b) Expansions of overlaid 2D 13 C- 13 C spectra of the U- 13 C, 15 N -syn fibril as prepared for solid-state NMR (red) and fibrils washed with HEPES buffer as the samples for electron microscopy (blue). Spectra were acquired at 600 MHz 1 H frequency, 13.3 khz magic-angle spinning (MAS) and 50 ms dipolar-assisted rotational resonance (DARR, Takegoshi, K., Nakamura, S. & Terao, T. 13 C- 1 H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem. Phys. Lett. 344, (2001).). (c) Equatorial intensity plot of the calculated and experimental fiber diffraction pattern of the -syn fibril. The solid line shows the experimental fiber diffraction pattern (equatorial intensity projected from Figure 4c) compared to the simulated pattern (dotted line) from the atomic coordinates of the structure presented in Figure 3. The simulated pattern recapitulates the main features of the experimental pattern including the fine features near 0.12 Å -1. The correlation coefficient between the two patterns is 0.77, in good agreement with previous amyloid fibril fiber diffraction comparisons.

7 Supplementary Figure 6 Comparison of the C chemical-shift perturbations for three early-onset Parkinson's disease mutant forms of -syn, relative to the wild-type chemical shifts. (a c) Chemical shift perturbation plots of the absolute deviation of the C shifts for A30P, E46K, and A53T previously published (Lemkau, L.R. et al. Mutant protein A30P α-synuclein adopts wild-type fibril structure, despite slower fibrillation kinetics. J. Biol. Chem. 287, (2012); Lemkau, L.R. et al. Site-specific perturbations of α-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K. PLoS One 8, e49750 (2013).) relative to the wild-type chemical shifts used in this work.

8 Supplementary Table 1. Solid-state nuclear magnetic resonance experiments Sample Experiment Magnet MAS Rate (khz) Mixing (ms) Exp Time (hr) A 2D 13 C- 13 C with DARR mixing 17.6 T (WB) A 2D 15 N-{ 13 CO}- 13 CX with DARR mixing 17.6 T (WB) A 2D 15 N-{ 13 CA}- 13 CX with DARR mixing 17.6 T (WB) A 2D 13 CA-{ 15 N}-{ 13 CO}- 13 CX with DARR mixing 17.6 T (WB) A 3D 13 CA-{ 15 N}- 13 CO 17.6 T (WB) N/A 4 A 3D 15 N- 13 CA- 13 CX with DARR mixing 17.6 T (WB) A 3D 15 N- 13 CO- 13 CX with DARR mixing 17.6 T (WB) B 3D 15 N- 13 CO- 13 CX with DARR mixing 11.7 T (WB) B 2D 13 C- 13 C with DARR mixing 11.7 T (WB) B 2D 13 C- 13 C with DARR mixing 11.7 T (WB) B 2D 15 N- 13 C with TEDOR mixing 14.1 T (WB) B 2D 15 N- 13 C with TEDOR mixing 14.1 T (WB) , 3.2, 4.8, 6.4, 9.6, 12.8, , 3.2, 4.8, 6.4, 8.0, 9.6, 12.8, , 13, 13, 9, 13, 13, 13 7, 14, 21, 35, 21, 21, 28, 28 B 2D 13 C- 13 C with DARR mixing 14.1 T (WB) B 3D 15 N- 13 CO- 13 CX with DARR mixing 14.1 T (WB) B 2D 13 C- 13 C with DARR mixing 14.1 T (WB) B 2D 13 C- 13 C with DARR mixing 14.1 T (WB) B 2D 13 C- 13 C with DARR mixing 17.6 T (NB) , 100, 200, 300, 400, , 100, 200, 300, 400, , 11, 12, 19, 33, 42 11, 6, 12, 13, 13, 14 B 3D 15 N- 13 CO- 13 CX with DARR mixing 17.6 T (NB) B 3D 15 N- 13 CO- 13 CX with DARR mixing 17.6 T (NB) B 3D 15 N- 13 CO- 13 CX with DARR mixing 17.6 T (NB) B 3D 15 N- 13 CO- 13 CX with DARR mixing 17.6 T (NB) C 3D 15 N- 13 CA- 13 CX with DARR mixing 11.7 T (WB) C 2D 13 C- 13 C with DARR mixing 14.1 T (WB) C 2D 15 N- 13 C with TEDOR mixing 14.1 T (WB) , 200, 300, 400, , 3.2, 4.8, 6.4, 9.6, 12.8, , 18, 18, 30, 48 17, 22, 30, 22, 30, 113 C 3D 15 N- 13 CA- 13 CX with DARR mixing 17.6 T (WB) C 3D 15 N- 13 CO- 13 CX with DARR mixing 17.6 T (WB) C 3D 13 CA- 15 N- 13 CO 17.6 T (WB) N/A 19 C 2D 13 C- 13 C with DARR mixing 17.6 T (WB) C 2D 13 C- 13 C with DARR mixing 17.6 T (WB) C 3D 13 C- 13 C- 13 C with DARR mixing 17.6 T (WB) , C 3D 15 N- 13 CA- 13 CX with DARR mixing 17.6 T (WB) C 3D 15 N- 13 CO- 13 CX with DARR mixing 17.6 T (WB) D 2D 13 C- 13 C with 1H-1H mixing 17.6 T (WB) D 2D 13 C- 13 C with DARR mixing 17.6 T (WB) E 2D 15 N- 13 C with TEDOR mixing 11.7 T (WB) F 2D 15 N- 13 C with TEDOR mixing 11.7 T (WB) Total Time 3049