Fig. 1: αB-AXA fibril resolved by cryo-EM.

a Sequence alignment of the distal region of the N-terminal domain of αB-crystallin showing conservation of the NT-IXI motif (boxed) across model species that were mutated in this work (top), and putatively identified NT-IXI regions in other related sHSPs (bottom). Residues colored by amino acid properties (non-polar—gray, aromatic—dark gray, polar—orange, basic—blue, acidic—red). In canonical IXI-motifs, isoleucine may be replaced by valine. In other sHSPs, such as HSPB1, the NT-IXI motif may be more cryptic, with isoleucine positions replaced by other hydrophobic residues. b Size-exclusion chromatography (SEC) profile of αB-wt (gray trace) and αB-AXA (blue trace). Peak fractions are labeled as (†), indicating region containing fibrils and (*) with native-like assemblies. c Representative EM micrographs (scale = 100 nm) isolated by SEC fractions (*, left) and (†, right), selected from a dataset of n = 119 and 93, respectively. Insets show representative 2D classes (n = 100). d Montage of 2D classes obtained by cryo-EM (scale = 10 nm), selected from n = 150 classes, to represent the fibrillar morphology. e Cryo-EM density map of the αB-AXA fibril state resolved at 3.4 Å resolution and (f). atomic model displayed in cartoon representation, with the ACD in blue, CTD in red and CT-IXI in orange. g Illustration of domain organization, colored as in panel (f) with NTD colored in gray (top), structure and illustration of the asymmetric unit showing 2-fold internal pseudosymmetry of the ACD dimer (middle), and schematic illustrating the internal D2 and helical symmetries present in the fibril assembly (bottom). h, i Zoomed views of CT-IXI motif binding with the ACD β4/8 groove at inner and outer seam, respectively. Cryo-EM density is displayed in transparency. Interacting residues between neighboring subunits are labeled.