The stage-wise macromolecular assembly and structure evolution of silk along the silk gland.

Silk-spinning organisms naturally produce fibers with extraordinary properties at minimal energy input. Silk protein (fibroin) feedstock's instability limits the mechanistic exploration of silk fiber formation to in-vitro studies of certain gland sections, offering a fragmented, non-native view of this process. Here, we unveil key events of fibroin stage-wise macromolecular assembly and structural transitions, essential for liquid feedstock storage and conversion into hierarchically structured solid fibers. By combining cryo-electron microscopy techniques, we monitor silk feedstock processing in-situ, at the nano-to-micron scales, along Bombyx mori silk gland and reveal nano-scale organizations. During storage, fibroin forms micron-sized spherical "compartments", ensuring protein stability and avoiding premature fibrillation. The spinning itself starts after the compartments' disassembly. It commences via a series of structural transitions, from protein alignment to forming several fibrillated nano-structures and, finally, a network of physically cross-linked nano-bundles, determining the final silk fiber structure and properties.