Real-time visualization of collagen assembly uncovers metastable properties in hierarchical organization

Collagen, a central component of the extracellular matrix (ECM), precisely regulates tissue mechanical properties and biological functions through hierarchical assembly, playing a vital role in maintaining homeostasis. However, the molecular mechanism of assembly remains poorly understood, limiting insights into tissue remodeling, aging, and ECM-related diseases. Here, we employ time-resolved cryo-electron microscopy to resolve two critical hierarchical intermediates in fibrillar collagen assembly, proposing the 3D collagen assembly pathway. We identify a metastable triple-helical conformation as the fundamental assembly unit, whose structural lability propagates through the assembly cascade, rendering the process sensitive to microenvironmental perturbations. Through hierarchical assembly, metastable intermediates achieve enhanced structural stability, culminating in the formation of stable collagen that retains its integrity under physiological conditions. In contrast, structural defects in intermediates lead to aberrant assembly and disruption of ECM integrity. Functional assays reveal that intermediates lacking D-band retain biological activity. Our findings redefine the fibrillar collagen assembly as a hierarchical, time-resolved cascade driven by metastable intermediates and propose the fundamental F-Z-F rules. The metastable triple helix provides a structural basis for hierarchical assembly and suggests a potential link between abnormal fibrillar collagen assembly and aging-related matrix dysfunction, offering valuable insights into collagen assembly and its role in aging diseases.