Figure 1. (a) Structural model representing a typical cross‐β arrangement. The fiber axis runs from down to up. Shown here is an antiparallel arrangement of six β‐strands. The carbonyl carbon of a given residue and the nitrogen atom of the partner forming an interstrand hydrogen bond, marked by dashed lines, are denoted by golden and green spheres, respectively. (b) Several layers of β‐sheets connected by hydrophobic interactions can form a twisted fiber (model based on register constraints obtained by NMR and an overall twist obtained by atomic force microscopy). (Part (a) adapted from [ 14], part (b) adapted from [ 88].)

Figure 2. – correlation spectrum DARR spectra (100 and 20 ms mixing, respectively) of the prion domains of HET‐s (a) and Ure2p (b) reveal the considerably higher order encountered in HET‐s(218–289) when compared to Ure2p1–93 spectrum. (HET‐s(218–289) adapted from [ 2], Ure2p1–93 adapted from [ 5].)

Figure 3. Rotor filling tool for ultracentrifuge. The rotor is inserted into an insert (here PEEK 30% glass) and a funnel is screwed directly onto the top of the rotor, which is then inserted into the bottom part of the assembly. The tool shown here fits into the buckets of a swinging rotor (Beckman SW 40 Ti) and was tested up to speeds of 35 000 rpm (SW 40 Ti) corresponding to 210 000 g in a Beckman Optima L‐90 K preparative ultracentrifuge. The tool shown fits a 3.2‐mm Bruker rotor. (Reproduced from [ 51].)

Figure 4. NCA two‐dimensional spectrum of HET‐s(218–289).

Figure 5. Three‐dimensional pulse sequences for assignment: schematic representation of the (a) NCACO, (b) NCOCA, and (c) CANCO pulse sequences. Adequate phase cycles are indicated to the right. The arrows with atom names symbolize the position to which the carrier frequency was set: CA corresponds to 56.5 ppm, CB to 42 ppm, and C′ to 176.5 ppm. The length of transfer periods and the maximum length of chemical shift evolution periods are indicated on top of each pulse sequence. (Adapted from [ 59].)

Figure 6. Representative planes from the three three‐dimensional spectra of HET‐s(1‐223) that contain a C′ dimension and that can alternatively be used for the backbone resonance assignment when resolution permits. All peaks correspond to sequential correlations picked in the spectra; the assignment is given where space permits. For two representative neighboring amino acids (V22, D23), the correlations used to establish sequential contact are highlighted in red. (a) δ2–δ3 plane from the NCACO spectrum at δ1 = 117.8 ppm. The spin system of V22 is highlighted. (b) δ2–δ3 plane from the NCOCA spectrum at δ1 = 117.1 ppm. The connection of the V22 C ^αC′ resonance pair to the N resonance of D23 is highlighted. (c) δ1–δ3 plane from the CANCO at δ2 = 117.1 ppm. The connection V22 C′ to the NC ^α resonance pair of D23 is highlighted. (Adapted from [ 59].)

Figure 7. Schematic representation of the pulse sequences NCACB, N(CO)CACB, CAN(CO)CB, N(CA)CBCX, and CCC (from a–e). Adequate phase cycles are indicated to the right. The arrows with atom names symbolize the position to which the carrier frequency was set: CA corresponds to 56.5 ppm, CB to 42 ppm, and C′ to 176.5 ppm. The lengths for transfer periods and the maximum lengths for chemical shift evolution periods are indicated on top of each pulse sequence. (Adapted from [ 59].)

Figure 8. Isotopic labeling strategies to distinguish between intra‐ and intermolecular restraints. The example shown (HET‐s) forms a β‐helix with two turns per molecule (the red part of the middle diagram represents one molecule). (Adapted from [ 14].)

Figure 9. Stacking models for cross‐β fibrils. In the figure, a molecule with two turns per molecule (two layers of β‐strands) is depicted, corresponding to the conformation already identified early‐on for HET‐s(218–289) [ 38]. For the parallel in‐register models (a) and (b), one molecule forms a unit cell, symbolized by a single Duplo block. Note that the top and bottom surfaces are twisted here as an example by an angle α = 4°. In (b), an additional 180̊ rotation around the fibril axis is added. For the antiparallel arrangements in (c) and (d), half of the Duplo blocks are rotated by a 180° rotation around either the x‐ or y‐axis (perpendicular to the fiber axis). This antiparallel arrangement defines two different types of molecular interfaces, which alternate along the fibril axis. The examples shown do not include a register shift, which may be added. (Adapted from [ 39].)

Figure 10. Extracts in the aliphatic and carbonyl region from two‐dimensional DARR spectra of Ure2p. The spectrum was recorded at a proton field strength of 850 MHz. (Adapted from [ 5].)