Fig 1.
Purification of L-type BSE prions.
(A) Silver stain SDS-PAGE gel of samples taken at different steps of the PTA purification. (B) Western blot of the L-type BSE prion purification samples using the D15.15 anti-prion monoclonal antibody. The PK-digested pellet 1 and the final pellet show the typical three bands of the bovine prion protein corresponding to di-, mono- and non-glycosylated forms (double arrowhead, single arrowhead, and open arrowhead, respectively). The two lanes labeled as final pellet represent the same sample. (C) Silver stain SDS-PAGE gel of samples from the sucrose step gradient centrifugation. The samples from the top, middle, bottom, and pellet wash fractions demonstrate the improved purity of the samples, even though the prion protein signal is relatively weak. Consistent with the Western blot analysis of the sucrose gradient fractions, the pellet-wash fraction shows stronger signals. (D) Western blot of the sucrose step gradient fractions. After ultracentrifugation through 40% and 80% sucrose, all fractions were separated based on their densities from top to bottom (A-L). The pellet-wash fraction (M) was obtained by washing the tube with 100 μl of sucrose buffer. The Western blotting was developed using the D15.15 antibody. As expected, the middle fraction (F) shows a stronger signal compared to the top (40%) and the bottom fractions (80%). The pellet-wash fraction (M) also showed a high yield of L-type BSE prions. The di-, mono-, and non-glycosylated bands are again indicated by a double arrowhead, single arrowhead, and open arrowhead, respectively. (E) Differences in migration patterns between L-type, C-type, and H-type BSE strains visualized in a Western blot of PK-treated brain homogenates of L-, C- and H-type BSE prions. Signals were detected using an anti-PrP monoclonal antibody, D15.15. In atypical BSE isolates, the electrophoretic migration of the unglycosylated PrP band is slightly higher (H-type) or lower (L-type) compared to classical BSE (C-type). The di-, mono-, and non-glycosylated bands are again indicated by a double arrowhead, single arrowhead, and open arrowhead, respectively.
Fig 2.
Negative stain electron micrographs of purified L-type BSE samples showing heterogeneous morphologies.
Representative electron micrographs of (A) large PrP 27–30 aggregates, (B) isolated forms of both single (arrowheads) and double (arrow) protofilament fibrils, and (C) 2D crystals along with amyloid fibrils. Grids stained with 2% uranyl acetate. Scale bar = 100 nm.
Fig 3.
Gallery of negatively stained L-type BSE fibrils.
Representative electron micrographs of L-type BSE fibrils with distinct morphologies containing two- (A) and one-protofilament (B) fibrils. The crossover regions for selected two-protofilament fibrils are shown using white arrows. Grids stained with 2% uranyl acetate. Scale bar = 100 nm.
Table 1.
Fibril dimensions of negatively stained brain-derived infectious L-BSE fibrils.
Fig 4.
Side-by-side comparisons of the one- and two-protofilament fibrils.
(A-F) Negative stain electron micrographs showing the two distinct morphologies of L-type BSE fibrils side-by-side. (A) A negative-stained electron micrograph showing a long, isolated fibril composed of two protofilaments (white arrowhead), which appears to be broken and split into two one-protofilament fibrils towards its lower end (black arrows). (B-F) L-type BSE amyloid fibrils showing a two-protofilament fibril at one end (white arrowheads) and two one-protofilament fibrils at the other end (black arrows). Scale bar = 100 nm.
Fig 5.
Width distribution of L-type BSE fibrils.
(A) A sample electron micrograph and histogram of the maximum width of 231 two-protofilament L-type BSE fibrils. (B) A sample electron micrograph and histogram of the maximum width of 636 one-protofilament L-type BSE fibrils. The width measurements were performed using EMAN’s boxer program.
Fig 6.
Alignment, classification, and averaging of L-type BSE fibrils.
(A-C) Reference-free alignment and classification of particles of two-protofilament L-type BSE fibrils. Class averages of 297 segmented particles exhibiting different regions of the fibrils, including the crossover region (A), showing two intertwined protofilaments with an apparent, stain-filled gap between them. (D-F) Class averages from the reference-free alignment of 532 particles of one-protofilament L-type BSE prion fibrils. All three class averages show a single helical filament only. Representative images of aligned fibril segments of one- (G) and (H) two-protofilament fibrils before classification.
Fig 7.
Three-dimensional reconstructions of L-type BSE prion fibrils.
(A) A 3D reconstruction of a two-protofilament fibril. (1) A negatively stained electron micrograph of a L-type BSE fibril, and (2) magnified view of the selected fibril section used for generating the 3D reconstruction. (3) A surface view of the 3D reconstruction showing two intertwined helical protofilaments. (4) Cross-section of the reconstruction. (5) The corresponding 2D projection of the averaged view of the cross-section region. (6) A contour map of the cross-section. (7) A contour density plot of the cross-section obtained by superimposition of the contour map onto a cross-section from the 3D volume. (B) A 3D reconstruction of a one-protofilament fibril. (1) A negatively stained electron micrograph of a L-type BSE fibril, and (2) enlarged view of the selected fibril. (3) Surface view of the 3D reconstruction. (4) Cross-section of the reconstruction. (5) The corresponding 2D projection of the averaged view of the cross-section region. (6) A contour map of the cross-section. (7) A contour density plot of the cross-section. The 3D reconstructions were low-pass filtered to 20 Å resolution. Scale bar = 10 nm.
Fig 8.
Gallery of independent, 3D reconstructions of two-protofilament L-type BSE fibrils.
(A) 3D volumes of different two-protofilament L-type BSE fibrils. (B) Cross-section views of the helical reconstructions. (C) Contour maps and (D) density maps of the reconstructed volumes. Scale bar = 10 nm.
Fig 9.
Gallery of independent, 3D reconstructions of one-protofilament L-type BSE fibrils.
(A) 3D volumes of various thin, one-protofilament L-type BSE fibrils. (B) Cross-section views of the helical reconstructions. (C) Contour maps and (D) density maps of the reconstructed volumes. Scale bar = 10 nm.
Fig 10.
Superimposition of the 3D L-type BSE fibril reconstructions with the proposed structures for PrPSc.
(A) A representative view of the 4RβS model of PrPSc with attached N-linked glycans superimposed onto the cross-section view of 3D reconstructions of a two-protofilament (red) and a one-protofilament (green) L-type BSE fibril. The size of the 4RβS model (50 Å x 30 Å) was set based on the previous cryo-EM and molecular dynamics simulation studies [24,25]. (B) A surface view of the 4RβS PrPSc model carrying glycans superimposed onto the cross-section view of 3D reconstructions of a two-protofilament (red) and a one-protofilament (green) L-type BSE fibril. (C) Top and (D) side views of a representative tetrameric version of the 4RβS PrPSc model containing glycans [25]. (E) Superimposition of the cross-section of a two-protofilament (red) and a one-protofilament (green) L-type BSE fibril and the PIRIBS structure obtained from the recent cryo-EM study on brain-derived 263K prions [28]. The size of the PIRIBS structure (13 nm) was set as indicated in the pre-print. The space-filling view shows the protein moiety only. Estimated densities representing N-linked glycans are attached to the structure in positions indicated in the pre-print [28].
Fig 11.
Immunogold electron microscopy of purified L-type BSE fibrils.
Decoration of different fibrillar assemblies, including (A) two-protofilament, (B) one-protofilament, and (C) fibrillar aggregates, with Fab 69 and a 5 nm gold-conjugated ternary detection system. White arrowheads highlight a few gold particles along the fibrils. (D) Grids that were incubated without primary antibody showed only rare gold particles (black arrow), demonstrating the specificity of the Fab 69 labeling. Immunogold labeling of (E) two-protofilament, (F) one-protofilament, and (G) aggregates of L-type BSE fibrils with Fab 29 and a 5 nm gold-conjugated ternary detection system. (H) No specific labeling was observed in the grids with no primary antibody. Scale bar = 100 nm.
Fig 12.
Immunogold labeling of purified L-type BSE fibrils with a conformation-dependent monoclonal antibody.
(A & B) Represents samples labeled with the YEG mAb Sc-G1 monoclonal antibody, which recognizes a discontinuous epitope on native PrPSc only. The monoclonal antibody was detected with a secondary antibody coupled to 6 nm gold particles (C) Control grid that underwent the same treatment except for the omission of the primary antibody, showing no gold labeling. Scale bar = 100 nm.
Table 2.
Infectivity of the purified L-type BSE samples.
Intracerebral inoculation of transgenic mice with the BSE prion samples obtained during purification experiments.