Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1. Identification of polymorphs and secondary structure.

Transcription

1 Supplementary Figure 1 Identification of polymorphs and secondary structure. (a) A comparison of the aliphatic spectral regions of 2D 13 C 13 C correlation spectra for non-seeded A (1 42) fibril (G 1 black) and A (1 42) fibril obtained by 3 successive seeded incubations (G 4 red) with slices along the indicated positions 1 4, which display cross peaks. The non-seeded fibril sample shows another set of cross peaks corresponding to the second conformer (black dotted circles) for Ala21, Val24, and Leu34 (assigned with blue arrows and labels). The seeded fibrils sample shows single set of cross peak for Phe20, Ala21, Val24 and Gly25 (assigned with black arrows and labels). Although Leu34 still displays a weak peak for the second set of cross peaks, it is clear that the secondary species was nearly completely suppressed by seeding. The base contour levels were set to (black) 5% and (red) 4% of the diagonal signal for 13 C α of Ala21, which both correspond to 4 times the root-mean-squared noise level. (b) Secondary 13 C chemical shifts for 13 C α (red), 13 C (blue), 13 CO (white) observed for the Aβ(1 42) fibrils by ssnmr. The secondary-shift value represents a deviation of the 13 C shift from that for the corresponding amino acid for a model peptide in a random coil conformation. The orange diamonds denote the residue that does not exhibit a combination of negative 13 C α and positive 13 C secondary shifts, which is typical for a -strand. (c) Dihedral angles (φ, ψ) obtained by TALOS-N analysis according to 13 C, 15 N, chemical shift analysis (Supplementary Table 1) of Aβ(1 42) in fibrils assemblies. The secondary structure analysis by the TALOS software indicated three extended β-strand regions (cyan arrows and cyan shadow) separated by loop/turn regions (black wave) at the residues of and as shown at the top of (b).

2 Supplementary Figure 2 Interstrand distance analyses and long-range intermolecular contacts in 2D 13 C- 13 C ssnmr. (a) Signal dephasing curves by fprfdr-ct experiments for determination of an inter-β-strand 13 CO 13 CO distance for seeded A (1 42) fibril sample (G 4 ), with 13 CO labeled at Ala30 (black circle) and Leu34 (green circle), respectively. The interstrand distances at Ala30 and Leu34 were both found to be 5.0 Å 0.1 Å. The results indicate an in-register parallel β-sheet arrangement. (b-d) Identification of inter-molecular cross peaks by a comparison of 2D 13 C- 13 C DARR spectra of (red) a 50%-isotope-labeled A (1 42) fibril sample and (black) a control sample made from 100% labeled A (1 42) for a 200-ms mixing time. The 50%-labeled seeded A (1 42) fibril sample (G 4 ) was prepared by incubating from a 1: 1 mixture of unlabeled A (1 42) peptide and A (1 42) that was labeled with uniformly 13 C-, 15 N-labeled amino acids at residue Gly29 and Ile41 (b), Phe19, Ala30, Ile31, Gly33, and Val36 (c), and Phe20, Ala21, Val24, Gly25, and Leu34 (d). The data were apodized with a Lorentz-to-Gauss window function with an inverse exponential of 50 Hz and a Gaussian broadening of 130 Hz in both the t 1 and t 2 domains. The base contour levels were at 4 5 times the root-mean-squared noise level; the base levels correspond to (b) 5%, (c) 12%, (d) 11% (black) and (b) 4%, (c) 11%, (d) 5% (red) of the diagonal signal for 13 C α of (b) Ile41, (c) Ala30, and (d) Ala21. The purple arrows in the slices indicate long-range intra-molecular cross peaks, for which 50% dilution of 13 C does not reduce the peak intensities more than 35%. Weak signals below the contour levels were not used for the quantitative analysis. The experimental time was (b) 3 and (c, d) 2 days for the 100% labeled sample, while that is (b) 4, (c) 2, and (d) 5 days for the 50% labeled sample.

3 Supplementary Figure 3 Overlaid ten best-fit atomic structure models of the A (1 42) fibril. The molecules at the edge and the N-terminal side chains at residues were omitted for clarity.

4 Supplementary Figure 4 A comparison of previously published structural models for A fibrils. (a c) Various atomic models of A (1 40) fibrils (top) for a single protofilament unit and (bottom) multiple units. (d) A hypothetical structural model for A (1 42) fibril. All the models were built from the pdb structures indicated in the figure. Experimentally observed side-chain contacts are denoted by orange dotted lines. All of the models share a U-shaped -loop- or -turn- structural motif with common contacts such as F19 L34 and D23 K28. For (d), side-chain contacts were deduced from pair-wise mutations, but no contacts were experimentally confirmed. PDB identifiers are indicated with the labels.

5 Supplementary Note and Tables Aβ(1 42) Fibril Structure Illuminates Self-recognition and Replication of Amyloid in Alzheimer s Disease Yiling Xiao 1), Buyong Ma 2), Dan McElheny 1), Sudhakar Parthasarathy 1), Fei Long 1), Minako Hoshi 3,4), Ruth Nussinov 2,5), Yoshitaka Ishii *1,6) 1 Department of Chemistry, University of Illinois at Chicago, Chicago IL Cancer and Inflammation Program, Leidos Biomedical Research, Inc., NCI-Frederick, Frederick, MD Institute of Biomedical Research and Innovation, Kobe , Japan. 4 Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Kyoto , Japan. 5 Sackler Inst. of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. 6 UIC Center for Structural Biology, University of Illinois at Chicago, Chicago IL Supplementary Note Protocols for solid-state NMR experiments in Supplementary Fig. 1 and 2 The 2D 13 C 13 C correlation spectra in Supplementary Fig. 1 were obtained with a fprfdr pulse sequence 1 using a mixing period of 1.6 ms at the spinning speed of 20,000 Hz ± 2 Hz, as discussed previously. 2 For the 2D data of the unseeded sample, a Lorenz-to-Gauss window function was applied with an inverse exponential narrowing (IEN) of 50 Hz and a Gaussian broadening (GB) of 80 Hz in the t 1 time domain while a Lorenz-to-Gauss function with IEN of 50 Hz and GB of 100 Hz was applied in the t 2 domain. The overall experimental time was 3 days. A similar apodization was used on the data for the seeded sample (G 4 ) with IEN of 45 Hz and GB of 80 Hz in both the t 1 and t 2 domains. The overall experimental time was 2 days. For the 1D 13 C- 13 C distance measurements in Supplementary Fig. 2a, we used a constant-time finite-pulse radio frequency-driven recoupling (fprfdr-ct) pulse sequence. 3 The spinning speed was 20,000 Hz ± 2 Hz. After the CP period of 1.0 ms, a train of rotor-synchronous 13 C π-pulses having a pulse width of 15 µs were applied during the mixing period with one π-pulse in a rotor period and the XY-16 phase cycle. The π-pulse trains were separated by π/2-pulses with a pulse width of 2.5 µs to achieve the constant-time dipolar dephasing based on WAHUHA-4 homonulear decoupling. The total mixing time was 14.4 ms and the effective mixing time was in a range of 0 12 ms. S1

6 Additional comparisons of the experimental shifts and the predicted shifts by ShiftX2 Supplementary Table 3 shows the deviation of the experimental 13 C and 15 N shifts from the corresponding shifts predicted by SHIFTX2 software (ver. 1.08) using the atomic-level structural model in Fig. 3 with a u option to include the effects of inter-strand interactions. The predicted shifts are ensemble average of the shifts of the listed 13 C or 15 N site for 12 Aβ molecules in the structural model. The root-mean-squared deviations (RMSD) between the experimental and predicted shifts were calculated for 13 C α, 13 C β, 13 CO, and 15 NH. The average RMSD value δ ave was obtained as a weighted average of the RMSD vales of 13 C α, 13 C β, 13 CO, and 15 NH δ j using " δ ave = $ # $ j % δ j /σ j ' &' / " $ # $ j 1/σ % 2 j ', where j = C α, C β, CO, and NH. The root-mean-square (RMS) errors σ j of the chemical &' shift prediction by SHIFTX2 for 13 C α, 13 C β, 13 CO, and 15 NH were 0.44 ppm, 0.52 ppm, 0.53 ppm, and 1.12 ppm, respectively. Although the RMSD values in Supplementary Table 3 are greater than the reported RMS errors, the obtained RMSD values are comparable to the corresponding RMSD values obtained for the amyloid-fibril structure for Het-s prion protein 4 (Supplementary Table 6; PDB: 2RMN). Considerable deviation between the experimental shifts and predicted shifts may be attributed partly to the bias of the prediction software using globular proteins for the prediction training. In particular, 15 N shifts deviates in a range of 3 4 ppm from the prediction for both of the current model in Fig. 3 and 2RNM. These top 10 models also best reproduced experimental 13 C shifts (Supplementary Table 5) among the 20 models selected in the AMBER analysis. The predicted shifts fit the experimental ones considerably better than those predicted for the published β-turn-β model (PDB: 2BEG) for the Aβ42 fibril (Table A1). Thus, we used the 10 models listed in Supplementary Table 5 as the best ensemble model (Supplementary Fig. 3). Although further optimization of the conformations are needed, the optimized atomic models reproduced experimental shifts reaonably well, confirming the validity of the anaysis. Additional information on the RMSD calculations for the 10 best-fit structures For the emsemble of the 10 best-fit models (Supplementary Table 5), the RMSD values of 1.00 Å and 1.47 Å from the mean structure were respectively obtained for the backbone and all the heavy atoms of the residues in the centeral Aβ molecule of the fibril models, suggesting a converged structural fold. Overlaid ensemble structures (Supplementary Fig. 3) present that all the 10 models show very similar tertiary folds except for a few sidechains near the loop regions and the dynamic N-terminus residues A relatively large RMSD value can be, in part, attributed to structural heterogeneity or dynamics in the turn regions. The corresponding RMSD values for the central four Aβ molecules are 1.08 Å and 1.53 Å for the backbone and all the heavy atoms, respectively; this suggests that a long-range translational symmetry can be further defined by complimentary methods such as cryoelectron microscopy. Although further refinements may be needed in our future work, all the models display important biological and structural features presented in this study. S2

7 Structural comparison to previously proposed structural models for Aβ(1 40) and Aβ(1 42) fibrils In previous studies using solid-state NMR, detailed structural models for Aβ(1 40) fibrils were presented Supplementary Fig. 4a c shows examples of high-resolution models reported in the Protein Data Bank. Although the study showed that structure of amyloid fibril depends not only on the amino-acid sequence, but also on the fibril growth condition, similar structural features were observed for its proto-filament unit of the Aβ(1 40) fibrils. The structural features of the present model for Aβ(1 42) fibril (Fig. 3), however, show a marked contrast to those of the previous Aβ(1 40) fibril as follows. (1) In Aβ(1 40) fibrils, the peptide commonly forms a hairpin structure with a β-turn-β structural motif, although different packing of the structural units and side-chain arrangements result in formation of different fibril morphologies. In the Aβ(1 42) fibril, we observed that three shorter β-strands are formed. This is consistent with a proposal of multiple β-strands based on previous prolinescanning studies of Aβ(1 42) fibrils, 11 although structurally heterogeneous fibril samples that were likely used in the study 12 make the data interpretation somewhat difficult. In our study, the data interpretation is straightforward because of our improved sample preparation. (2) In the Aβ(1 40) fibrils, Lys28 side chain forms a salt bridge with that of Asp23, which substantially stabilizes the hairpin structure. A recent study using Asp23 Lys28 lactam Aβ(1 40) showed that the formation of the salt bridge in monomeric Aβ(1 40) promotes fibril formation, 13 suggesting that this salt bridge may be formed at an early stage of misfolding for Aβ(1 40). For the Aβ(1 42) fibril, our study has revealed that Lys28 forms a salt bridge with carboxyl terminus of the Ala42 residue, which is missing in Aβ(1 40). The results suggest that misfolding path of Aβ(1 42) is likely different from the monomeric or small oligomeric state. (3) Asp23 does not form a salt bridge with any other residues in Aβ(1 42). Both Asp23 and Glu22 are pointing to the outside of the hydrophobic core of the fibril, forming a part of the loop region. (4) When an amyloid protein forms multiple inter-molecular β-sheets in the sequence along the fiber axis, displacement of a β-strand segment along fiber axis is another structural parameter. This displacement called staggering was reported for Aβ(1 40) fibril. Staggering of the β-sheet chain is minimal in the Aβ(1 42) fibril. Most of the long-range contacts were assigned to intra-molecular contacts. So far, the packing of multiple protofilament units is not known for the Aβ(1 42) fibrils unlike the Aβ(1 40) fibril models (Supplementary Fig. 4a-c). Our STEM images (Fig. 3d) showed fibrils with a periodic modulation in the diameter between 6 ± 1 nm and 13 ± 1 nm. The STEM data are likely to indicate bundling of multiple basic proto-filament units shown in Fig. 3. Fibril species with similar dimensions were reported in previous electron micrograph studies of Aβ(1 42) fibrils, 14,15 which suggest bundling of two or more proto-filament units based on STEM or cryoem data. Our STEM data also showed another species of thinner filaments with a modulation approximately between 4.5 and 6.0 nm. The proposed model also shows distinctive structural features from the hypothetical model obtained for amyloid fibril of the M35L-mutant of Aβ(1 42) 16 (Supplementary Fig. 4d). This work is based on an assumption that Aβ(1 42) fibrils retains (i) the same parallel β-sheet structure and (ii) a salt bridge between Asp23 and Lys28 observed for Aβ(1 40) fibrils and other types of Aβ(1 42) fibrils, the latter of which could be fundamentally S3

8 incorrect. Also, at the early stage of amyloid structural studies, it was assumed that the sample was structurally homogeneous without a sufficient experimental basis. Such an assumption may result in a misleading structure from the H/D exchange data if the heterogeneity is substantial. Nevertheless, this model well explains pair-wise mutation results. Our atomic-level structural model in Fig. 3 shows marked differences from the model, including previously unexpected structural features such as the salt-bridge formation between Ala42 and Lys28 and the unique triple β-sheet motif. Although the structural differences can be attributed to the presence of other polymorph(s), our atomic model in Fig. 3 better explains kinetic features of Aβ(1 42) fibril, which do not easily introduce cross-propagation of Aβ(1 40). Recent solid-state NMR structural studies of Aβ(1 42) fibrils indicated long-range contacts such as Phe19 Leu34 and Met35 Ala42, which are not indicated in our atomic model. 17,18 As indicated in Supplementary Fig. 1, alternate folds or polymorphs are likely to be present for Aβ(1 42) fibrils. Further studies are needed to fully define structures and structural variations of Aβ(1 42) fibrils. S4

9 Table A1. Deviation of calculated chemical shifts from experimental value for the earlier model of Aβ(1 42) fibril (PDB ID: 2BEG). (Experimental shift Simulated shift) Residue C α C β CO NH V F F A D V G S K G A I I G L V G G V V I Average RMSD a) Average b) C α C β CO NH a) The experimental shifts and the structure were obtained from ref. 16. The 50 Aβ structures in the pdb file were used for the calculation. The RMSD values were obtained from the average shifts. b) The average RMSD value was calculated as a weighted average as defined in Supplementary Table 3. S5

10 Supplementary Tables Supplementary Table C, 15 N chemical shifts and predicted torsion angles of Aβ(1 42) in seeded fibrils. Residue Chemical shifts (ppm) a) Torsion angles (degree) b) CO C α C β C γ C δ C ε C ζ C γ2 C δ2 NH N ζ A E3-72±12* 130±12* F G V ±13 139±10 H ±14 140±14 H ±22 150±19 L ±13 128±8 V ±7 129±8 F c) ±16* 130±11* F ±19* 145±15* A ±15 148±16 E22-66±8 146±11 D ±16* 141±14* V ±11 148±12 G ±51* 169±79* S ±16 156±10 N27-70±14 129±11 K ±15 139±12 G ±40* 170±29* A ±22 139±13 I ±12 138±11 I ±15 139±12 G ±21* 157±9* L ±8-29±9 M ±16* 126±14* V ±12 134±12 G ±78* -123±66* G ±11* 32±16* V ±12 140±15 V ±12 130±8 I ±10 126±6 A a) 13 C shifts were referenced to neat TMS reference, which is off from the DSS reference by 2.01 ppm. 19,20 15 N shifts were referenced to liquid NH b) Elucidated from TALOS-N analysis. The angles marked by * were predicted with Warning or as Dynamic regions, and these angles were not used for modeling. c) Partially overlapping with the shoulder of F19 Cε and Cδ peaks Φ ψ S6

11 Supplementary Table 2. Structural restraints from solid-state NMR experiments for the Aβ(1 42) fibrils. Type of contact Contacts or restraints identified Number of restraints Torsion angles (φ, ψ) E3, V12, H13, H14, L17-I41 38 Contacts between neighboring residues Long-range contacts a) (Intra-molecule) Long-range contacts c) (Inter-molecule) Missing long-range contacts from DARR experiments (intra- or inter-molecule) e) F20-A21, V24-G25, A30-I31, G33-L34 F19C δ/ε A30C α, F19C δ/ε A30C β, F19C δ/ε I31C γ2, F19C δ/ε I31C δ, F19C δ/ε I32C γ1, F19C δ/ε I32C γ2, F19C δ/ε I32C δ, F20C ε V24C γ1, b) K28N ζ A42CO, G29CO I41C δ, G29C α I41C δ, G29CO I41C γ2, G29CO I41C γ1, G29CO I41C α, G29C α I41C α, G29C α I41C β, G29C α I41C γ1, G29C α I41C γ2, A30CO A30CO, L34CO L34CO L17 A21, L17 G29, L17 L34, L17 G38, V18 D23, V18 S26, V18 K28, V18 I32, F19 G33, F19 L34, F19 V36, F19 G38, F19 V40, F20 G25, F20 L34, F20 V39, F20 I41, A21 V24, A21 G25, A21 G29, A21 L34, D23 S26, D23 K28, V24 L34, G25 L34, S26 K28, A30 I32, A30 G33, A30 V40, I31 G33, I31 L34, I31 V36, I31 G38, I31 V39, I31 V40, I32 G38, I32 V40, G33 V36, L34 G38, L34 V40, V36 G38, G38 V40, V39 I41 62 of total restraints 11 9 (2) d) 43 (27) f) a) F19Cδ and F19Cε are indistinguishable as these peaks overlap. b) This peak is weaker, but above the noise level. c) All are likely attributed to contacts between neighboring hydrogen-bonded Aβ molecules. d) The inter-molecular G29 I41 contacts were not used as structural restraints. e) Missing contacts are restricted to well-ordered residues measured for the samples that have sufficient high sensitivity in long-range DARR experiments. f) Only side chain contacts for the 27 pairs were used as structural restraints. S7

12 Supplementary Table 3. Deviation of the calculated chemical shifts by SHIFTX2 using the structural model in Fig. 3 from experimental values. Residue (Experimental shift Simulated shift) (ppm) 13 C α 13 C β 13 CO 15 NH V H H L V F F A D V G S K G A I I G L V G G V V I RMSD shifts a) Average b) 13 Cα 13 Cβ 13 CO 15 NH a) RMSD shifts δ j denote root-mean-squared deviation between experimental and predicted shifts for 13 C α, 13 C β, 13 CO, or 15 NH shifts, where j = 13 C α, 13 C β, 13 CO, or 15 NH. b) The average RMSD value δ ave was obtained as a weighted average of the RMSD vales of 13 C α, 13 C β, 13 CO, 15 NH " % using δave = $ δ j /σ j ' # $ j &' / " 1/σ % 2 $ j ', where j = C α, C β, CO, NH. The standard deviation values of σ Cα, σ Cβ, σ CO, σ NH # $ j &' were the root-mean-square errors of the chemical shift prediction of 13 C α, 13 C β, 13 CO, 15 NH as discussed above. 21 S8

13 Supplementary Table 4. Labeled samples used in this study. Sample Number a) Labeling Scheme b) 1* # F20, A21, V24, G25, L34 2 # A2, G9, F20, V39, I41 3 # F4, V12, L17, A21, G29 4 H13, V18, I32, G37 5 # V18, D23, S26, K28 6* # F19, A30, I31, G33, V36 7 # F19, A30, I32, G38, V40 8 # F19, I31, L34, G38, V40 9 F19, G33, L34, V36, A42 10 A21( 15 N), A30( 13 CO) 11 L17( 15 N), L34( 13 CO) 12 # I31, V39 13* # G29, I41 14 L17, I31, L34, V36 15* K28( 15 N), A42 16 # I31( 13 CO), G33, M35( 13 C ε ), V36( 15 N) 17 # H14, L17, V36, G38 a) For the samples denoted by *, 50% isotope labeled Aβ(1 42) fibril sample was also prepared by incubating a 1: 1 mixture of unlabeled Aβ(1 42) peptide and 13 C- and 15 N-labeled Aβ(1 42) peptide, besides 100% isotope labeled Aβ samples. b) Unless otherwise mentioned, 13 C-, 15 N-uniformly labeled amino acids were introduced at residue noted above. c) Samples used for the long-range DARR experiments are indicated by #. S9

14 Supplementary Table 5. Summary of the energies and chemical-shift RMSD values for 10 best-fit Aβ(1 42) fibril models obtained in the present study. Mol ID in pdb a) RMSD from the mean structures (Å) b) AMBER energy rank a) AMBER energy (kcal/mo le) CYANA energy rank c) 13 C α 13 C β RMSD chemical shift (ppm) 13 CO 15 NH Average RMSD d) Average RMSD for 13 C d) 1 e) a) Selected from the 100 structures tested by AMBER as described in the Methods. The 10 structures were selected by filtering the models that best reproduce 13 C and 15 N chemical shifts among the 20 structures obtained by AMBER energy minimization including the energies for penalty functions for structural restraints. The Mol ID is the ID number in the pdb file. b) The RMSD values of the backbone atomic positions from the mean of the 10 structures listed here. The central 4 Aβ was used for the RMSD calculation. c) The CYANA energy rank in the first stage screening. d) The average RMSD shifts were obtained as defined in Supplementary Table 3. e) This model shown in Fig. 3 was selected as the best representative model as the RMSD deviation is smallest to the mean structure. The structure also well reproduces torsion-angle and distance restraints. S10

15 Supplementary Table 6. The chemical-shift RMSD values for Het-s prion fibril structure obtained by solid-state NMR. Average b) RMSD chemical shifts a) (ppm) 13 C α 13 C β 13 CO 15 NH a) The experimental shifts and the structure were obtained from ref. 4. The first model of the 20 ensemble structures in the pdb file was used for the calculation. The RMSD values were obtained from the average shifts of the five Het-s protein molecules forming the fibril model. b) The average shift was calculated as defined in Supplementary Table 3. Supplementary Table 7. Summary of Ramachandran statistics and additional distance and dihedral statistics for the best-fit and ensemble Aβ(1 42) fibril models. Ramachandran regions a) Best-fit model (Fig. 3) Average of the 10 models in Supplementary Table 5 Most favored regions 84.0% 89.1% Additionally allowed regions 12.7% 10.8% Generously allowed regions 3.3% 0.1% Disallowed regions 0.0% 0.0% Distance violations / Aβ molecule b Å Å > 0.5 Å Dihedral angle violations / Aβ molecule b > a) Reported by Procheck in the PSVS software. b) The number of violations in this range per Aβ molecule. S11

16 Supplementary Table 8. Extended β-strand regions calculated by Stride software a) in the central 4 Aβ molecules (chain E-H) in the atomic model. Mol ID # Residues in the first extended β-strand region (Average β-region) b) Residues in the second extended β-strand region 24-33, 24-33, 24-33, (24-33) 24-32, 24-32, 24-32, (24-32) 24-33, 27-33, 27-33, (26-33) Residues in the third extended β-strand region 36-40, 36-40, 36-40, (36-40) 36-40, 36-40, 36-40, (36-40) 36-40, 36-40, 36-40, (36-40) 24-35, 24-35, 24-35, (24-35) c) 36-40, 36-40, 36-40, (36-40) c) 24-32, 24-32, 24-32, (24-32) 24-33, 24-33, 24-33, (24-33) 24-33, 24-33, 24-33, (24-33) 36-40, 36-40, 36-40, (36-39) 36-40, 36-40, 36-40, (36-40) 36-40, 36-40, 36-40, (36-40) 24-35, 24-35, 24-35, (24-35) c) 36-40, 36-40, 36-40, (36-40) c) 24-33, 24-33, 24-33, (24-33) 36-40, 36-40, 36-40, (36-40) 24-32, 24-32, 24-35, , 35-40, 36-40, (24-34) c) (36-40) c) Average a) The Stride software is available at b) The average region was obtained by rounding the average of the initial or final residue numbers of the region after manual alignment to maximize the overlapped regions. c) The extended second β-sheets ranging from residue 24 to residue 40 for Mol ID 4, 8, and 10 were split into two at the residue 36. S12

17 References: 1. Ishii, Y. 13C-13C dipolar recoupling under very fast magic angle spinning in solid-state NMR: Applications to distance measurements, spectral assignments, and high-throughput secondary-structure elucidation. J. Chem. Phys. 114, (2001). 2. Chimon, S. et al. Evidence of fibril-like ß-sheet structures in neurotoxic amyloid intermediate for Alzheimer's ß-amyloid. Nat. Struct. Mol. Biol. 14, (2007). 3. Ishii, Y., Balbach, J.J. & Tycko, R. Measurement of dipole-coupled lineshapes in a many-spin system by constant-time two-dimensional solid state NMR with high-speed magic-angle spinning. Chemical Physics 266, (2001). 4. Wasmer, C. et al. Amyloid fibrils of the HET-s( ) prion form a beta solenoid with a triangular hydrophobic core. Science 319, (2008). 5. Lu, J.-X. et al. Molecular Structure of β-amyloid Fibrils in Alzheimer s Disease Brain Tissue. Cell 154, (2013). 6. Petkova, A. et al. A structural model for Alzheimer's b-amyloid peptide fibrils based on experimental constraints from solid-state NMR spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 99, (2002). 7. Petkova, A.T., Yau, W.M. & Tycko, R. Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils. Biochemistry 45, (2006). 8. Paravastu, A.K., Leapman, R.D., Yau, W.M. & Tycko, R. Molecular structural basis for polymorphism in Alzheimer's beta-amyloid fibrils. Proc. Natl. Acad. Sci. U. S. A. 105, (2008). 9. Bertini, I., Gonnelli, L., Luchinat, C., Mao, J. & Nesi, A. A New Structural Model of Aβ40 Fibrils. Journal of the American Chemical Society 133, (2011). 10. Lopez del Amo, J.M. et al. An Asymmetric Dimer as the Basic Subunit in Alzheimer's Disease Amyloid beta Fibrils. Angew. Chem. Int. Edit. 51, (2012). 11. Morimoto, A. et al. Analysis of the secondary structure of beta-amyloid (A beta 42) fibrils by systematic proline replacement. J. Biol. Chem. 279, (2004). 12. Masuda, Y. et al. Identification of Physiological and Toxic Conformations in A beta 42 Aggregates. Chembiochem 10, (2009). 13. Sciarretta, K.L., Gordon, D.J., Petkova, A.T., Tycko, R. & Meredith, S.C. A beta 40-Lactam(D23/K28) models a conformation highly favorable for nucleation of amyloid. Biochemistry 44, (2005). 14. Antzutkin, O.N., Leapman, R.D., Balbach, J.J. & Tycko, R. Supramolecular structural constraints on Alzheimer's beta-amyloid fibrils from electron microscopy and solid-state nuclear magnetic resonance. Biochemistry 41, (2002). 15. Schmidt, M. et al. Comparison of Alzheimer A beta(1-40) and A beta(1-42) amyloid fibrils reveals similar protofilament structures. Proc. Natl. Acad. Sci. U. S. A. 106, (2009). 16. Luhrs, T. et al. 3D structure of Alzheimer's amyloid-beta(1-42) fibrils. Proc. Natl. Acad. Sci. U. S. A. 102, (2005). 17. Ahmed, M. et al. Structural conversion of neurotoxic amyloid-beta(1-42) oligomers to fibrils. Nat. Struct. Mol. Biol. 17, (2010). 18. Murakami, K., Masuda, Y., Shirasawa, T., Shimizu, T. & Irie, K. The turn formation at positions 22 and 23 in the 42-mer amyloid beta peptide: The emerging role in the pathogenesis of Alzheimer's disease. Geriatrics & Gerontology International 10, S169-S179 (2010). 19. Morcombe, C.R. & Zilm, K.W. Chemical shift referencing in MAS solid state NMR. J. Magn. Reson. 162, (2003). 20. Harris, R.K. et al. Further conventions for NMR shielding and chemical shifts (IUPAC recommendations 2008). Pure and Applied Chemistry 80, (2008). 21. Han, B., Liu, Y., Ginzinger, S.W. & Wishart, D.S. SHIFTX2: significantly improved protein chemical shift prediction. J. Biomol. NMR 50, (2011). S13