Subcellular membrane-less organelles consist of proteins with low complexity domains. Many of them, such as hnRNPA1, can assemble into both a polydisperse liquid phase and an ordered solid phase of amyloid fibril. The former mirrors biological granule assembly, while the latter is usually associated with neurodegenerative disease. Here, we observe a reversible amyloid formation of hnRNPA1 that synchronizes with liquid–liquid phase separation, regulates the fluidity and mobility of the liquid-like droplets, and facilitates the recruitment of hnRNPA1 into stress granules. We identify the reversible amyloid-forming cores of hnRNPA1 (named hnRACs). The atomic structures of hnRACs reveal a distinct feature of stacking Asp residues, which contributes to fibril reversibility and explains the irreversible pathological fibril formation caused by the Asp mutations identified in familial ALS. Our work characterizes the structural diversity and heterogeneity of reversible amyloid fibrils and illuminates the biological function of reversible amyloid formation in protein phase separation.

Significance: Posttranslational modifications (PTMs) play an important role in modulating α-syn structure, function, and pathology. Phosphorylation commonly applies to pathological α-syn aggregation in Lewy bodies and is a pathological hallmark of Parkinson’s disease (PD). Here, we obtain homogeneous α-syn fibrils formed by semisynthesized α-syn with site-specific phosphorylation at Y39. The synthetic fibril recapitulates the neuropathology and degradation resistance of endogenous pY39 α-syn aggregation in PD. Cryo-EM structure determination of the pY39 α-syn fibril reveals a fold of α-syn with the entire N terminus involved in the fibril core attracted by pY39. This structure illuminates the mechanism of pY39 pathology and highlights the importance of PTMs in defining the structure and pathology of amyloid aggregation in neurodegenerative diseases.