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 Å 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 ␤-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.electron microscopy ͉ image processing ͉ Nanogold labeling ͉ parallel ␤-helix ͉ amyloid structure C reutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie, and other spongiform encephalopathies are caused by an aberrantly folded isoform (PrP Sc ) of the prion protein (PrP) (1). Replication of prions includes a profound change in the conformation of the cellular isoform of PrP (PrP C ) to form the highly insoluble PrP Sc . The insolubility of PrP Sc has thwarted attempts to investigate its structure by either x-ray crystallography or NMR spectroscopy. Our knowledge about the structure of PrP Sc is therefore rather limited (2).After treatment with proteinase K (PK), PrP Sc loses the N-terminal residues 23 to Ϸ89 (forming PrP 27-30), but retains infectivity. During purification, PrP 27-30 polymerizes into rod-shaped filaments with the tinctorial properties of amyloid (3, 4). X-ray fibril diffraction illustrated the amyloid nature of PrP 27-30; characteristic 4.7 Å reflections indicative of cross-␤ structure were observed (5). Optical spectroscopy revealed that PrP Sc and PrP 27-30 are substantially enriched in ␤-sheet structure (6-9). This finding is in sharp contrast to the predominantly ␣-helical fold of the three-helix-bundle structure of PrP C as determined by NMR spectroscopy and x-ray crystallography on refolded recombinant PrP (10 -18). Owing to the lack of high-resolution structural information for PrP Sc , predictive methods have been used to develop molecular models to codify the existing spectroscopic, immunological, and biochemical data (19).In attempts to simplify the structural analysis of PrP Sc , we systematically deleted parts of the prion protein. One of these constructs containing only 106 residues, PrP106 (⌬23-88, ⌬141-176), supported the propagation of prions (20,21). Transgenic mice expressing only PrP106 develop a histologically accurate neurodegenerative prion disease after inoculation with prions, and the resulting prions can...

The gamma-tubulin ring complex (gammaTuRC) is a protein complex of relative molecular mass approximately 2.2 x 10(6) that nucleates microtubules at the centrosome. Here we use electron-microscopic tomography and metal shadowing to examine the structure of isolated Drosophila gammaTuRCs and the ends of microtubules nucleated by gammaTuRCs and by centrosomes. We show that the gammaTuRC is a lockwasher-like structure made up of repeating subunits, topped asymmetrically with a cap. A similar capped ring is also visible at one end of microtubules grown from isolated gammaTuRCs and from centrosomes. Antibodies against gamma-tubulin label microtubule ends, but not walls, in centrosomes. These data are consistent with a template-mediated mechanism for microtubule nucleation by the gammaTuRC.

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