STEM mass measurements
To study the molecular packing of β2m into this homopolymer, STEM was used to determine mass per unit length (MPL) along the fibrils. The results revealed a major component at 53 ± 3 kDa/nm, as well as fibrils with half the width and a component at 27 ± 3 kDa/nm (Fig. 5). The former is consistent with the A-type and B-type fibrils, while the latter presumably represents the C-type fibrils. The component at 62 ± 3 kDa/nm may correspond to more tightly twisted fibrils, and minor components at higher mass may correspond to different fibril morphologies. The mass measurements of the major component indicate that there are 24 β2m monomers (monomer mass, 11.8 kDa) in each 5.25-nm repeat of the whole B-type fibril, or four monomers in each repeat of each protofilament. The alternating wide and narrow interfaces between density features along the protofilaments (Fig. 4a and b) suggest that the bi-lobed density corresponds to a head-to-head arrangement of each assembly unit. This tetrameric unit has 2-fold symmetry rather than 4-fold symmetry, so that the building block of the protofilament comprises four β2m molecules in a dimer-of-dimers arrangement.
The hierarchical fibril structure is explained schematically in Fig. 6. Three bi-lobed density units assemble into an extended crescent shape. Pairs of these crescent-shaped elements join back to back to form one layer of the fibril structure. Many copies of this assembly stack to form the slowly twisting helical fibril, in which columns of the globular building blocks constitute the six protofilaments of the fibril. In contrast to the generally accepted notion of a continuous cross-β core in amyloid fibrils,4,6 the globular substructure of these protofilaments gives them the appearance of a string of bi-lobed beads. The polarity of the half-fibrils arises from asymmetric connections between the protofilaments in the crescents.