Cryo-ET comparison of the hierarchical ultrastructure of silkworm, spider, and artificial silk fibers.

Spider and silkworm silks are renowned for their exceptional mechanical properties, which arises from their ultrastructural organization. However, this architecture remains incompletely understood. Here, we apply cryo-electron tomography to examine the hierarchical organization of silkworm, spider, and artificial silks. In silkworm silk, we observe nanofibrils of ~3.6 nm in diameter, interconnected by abundant bridges and representing the smallest fibrillar features currently accessible by cryo-ET. These nanofibrils align with the fiber axis and are organized into a herringbone pattern, with stacked layers building the micron-scale filament. Spider silk displays densely packed nanofibrils with near-perfect axial alignment and minimal voids. In contrast, silkworm silk shows regionally heterogeneous gaps, whereas artificial silk lacks the ordered packing characteristic of natural materials. These observations provide a structural basis for understanding silk formation and may guide future biomimetic fiber design.