Direct cryo-EM visualization of the β-sheet structure in curved amyloid protofibril

Protofibrils are key intermediates to explore effective therapeutic strategies for amyloid-related diseases; however, their structural features remain largely ambiguous. Here, we report a direct visualization of the intermolecular β-sheet structure of amyloid protofibrils using cryo-electron microscopy. We analyzed the protofibrils formed by an insulin-derived peptide and observed 4.7 Å regularly spaced lines at their centers surrounded by fuzzy regions, consistent with the hydrogen-bonded β-strand structure. We propose a model in which short β-strands form a β-sheet core in the center, whereas the outer fuzzy regions are composed of random coiled structures. This structure is different from that of mature amyloid fibrils, where β-sheets span the entire structure, suggesting that the partial β-sheet formation in protofibrils is responsible for their curvy noodle-like appearance. Overall, this study highlights cryo-electron microscopy as a powerful tool for visualizing seemingly flexible structures, such as protofibrils, and establishes a conceptual framework for the rational design of diagnostic and therapeutic agents targeting the protofibril β-sheet structure.