Structural studies of the scrapie prion protein by electron crystallography - PubMed (original) (raw)

Structural studies of the scrapie prion protein by electron crystallography

Holger Wille et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Because the insolubility of the scrapie prion protein (PrP(Sc)) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP(Sc) (PrP 27-30) and a miniprion (PrP(Sc)106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 A resolution. By comparing projection maps of PrP 27-30 and PrP(Sc)106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP(Sc). Only models featuring parallel beta-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.

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Figures

Figure 1

Figure 1

2D crystals of PrP 27-30. (A) A 2D crystal of PrP 27-30 stained with 2% uranyl acetate showing an apparent hexagonal lattice. (B) High power view of a crystal after CTF correction and several rounds of correlation-mapping and averaging. (C) Section of a power spectrum after averaging showing spots out to the 11th order, corresponding to ≈7 Å (arrow). (D) Crystallographic averaging further improved the amount of detail visible. A p3 plane group was used. (E) Typical prion rod with an aggregate of “crystal” subunits at each end. Some protofilaments reveal rows of dense stain accumulations, suggesting stacked subunits (arrowheads). [Bars = 100 nm.]

Figure 2

Figure 2

Nanogold labeling of the N-linked sugars. (A) Uranyl acetate-stained 2D crystal of Nanogold-labeled PrP 27-30. The high contrast of the uranyl stain obscures some of the labels, but others are clearly visible (arrowheads). [Bar = 100 nm.] (B) Image processing result of a labeled crystal after correlation-mapping and averaging followed by crystallographic averaging. (C) Subtraction map between labeled and unlabeled crystals showing major differences in lighter shades. (D) Overlay of the statistically significant differences calculated from C in yellow onto a projection map of PrP 27-30.

Figure 3

Figure 3

2D crystals of PrPSc106. (A) A 2D crystal of PrPSc106 stained with uranyl acetate. [Bar = 100 nm.] (B) Image processing result after correlation-mapping and averaging followed by crystallographic averaging. (C and D) Subtraction maps between the averages of PrP 27-30 (Fig. 1_D_) and PrPSc106 (B). (C) PrPSc106 minus PrP 27-30 and (D) PrP 27-30 minus PrPSc106, showing major differences in lighter shades. (E and F) The statistically significant differences between PrP 27-30 and PrPSc106 calculated from C and D in red and blue, respectively, overlaid onto the crystallographic average of PrP 27-30 (Fig. 1_D_).

Figure 4

Figure 4

β-Helical models of PrP 27-30. (A and B) Top and side views, respectively, of PrP 27-30 modeled with a left-handed β-helix. The β-helical portion of the model is based on the Methanosarcina thermophila γ-carbonic anhydrase structure. (C and D) Top and side views, respectively, of the trimer of dimer model of PrP 27-30 with left-handed β-helices. (E and F) Top and side views, respectively, of PrP 27-30 modeled with a right-handed β-helix. The β-helical portion of the model is based on the most regular helical turns of Bordetella pertussis P.69 pertactin. (G and H) Top and side views, respectively, of the trimer model of PrP 27-30 with right-handed β-helices. The structure of the α-helices was derived from the solution structure of recombinant hamster PrP (–13). In the single-molecule images (A, B, E, and F), residues 141–176 that are deleted in PrP106 are colored blue.

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