High-resolution structure and strain comparison of infectious mammalian prions, Kraus et al.
This dataset includes image files associated with the manuscript "High-resolution structure and strain comparison of infectious mammalian prions", Kraus et al., Molecular Cell 2021. Corresponding Figure Legends: Fig. 1. EM images and density maps of 263K prion fibrils. (A) Negatively stained transmission EM image (bar = 50 nm). (B) Raw 2D cryo-EM image. Fig. 3. Anchorless RML (aRML) prions have distinct fibril topologies and cross-sectional densities. (A) Negative stain electron micrograph of aRML fibril preparations. Scale bar = 50 nm. (B) Representative micrograph of cryo fibril preparations. Scale bar = 50 nm. Supplemental Fig. 1. 263K PrPSc preparations are protease-resistant, seed-competent, and highly infectious. (A) Gel analysis of two independent hamster 263K purifications. Purified 263K prions from two preparations were subjected to PK digestion (PK+) and used for gel analysis and subsequent silver staining or PrP immunoblotting as indicated. Untreated 263K (PK-) is shown in the first lane. Predominant bands at ~20-32 kDa, or oligomers thereof, comigrated with bands reacting with PrP antibodies in the immunoblot. PK-generated size shift was consistent with cleavages within the N-terminal domain up to ~residue 90, leaving the remaining C-terminal residues intact (Hope et al., 1986; Prusiner et al., 1984). Densitometry of protein-stained gels indicated that the preparations were ~97-98% PrP. (E) Brain tissue from animals inoculated at the highest dose was used for PrP and GFAP immunostaining, and hematoxylin and eosin (H&E) staining. Consistent with the features observed with typical 263K clinical disease, PrP deposition, astrogliosis (GFAP), and spongiform change were observed. Scale bar = 50 µm Supplemental Figure 2. Tomography of 263K prions shows left-handed fibrils and asymmetric decoration with globules. (A) Tomographic slice of a fibril with globules visible on the ends, outlined with white circles in (A’). The globules were spaced at 7-10 nm but could be seen along the entire length of some fibrils (Supplemental Fig. 1A,B & Movies S1-3) and increased overall fibril widths by ~2-4 nm (Supplemental Table 1). Although the nature of these globules remains unclear, they might have been due in part to binding of residual lipids, detergent or other non-PrPSc molecules. (B) Another slice with globules, as marked in (B’). (C,D) Two slices of a 263K fibril without apparent globules. (E-H) Fibrils from 4 different tomograms showing the top, middle, and bottom slices, illustrating the left-handed helix found in >99% of fibrils analyzed by tomography. Scale bars: (A-D) = 25 nm, (E-H) = 50 nm.