Sanchez, J. C.; Pierson, J. A.; Borcik, C. G.; Rienstra, C. M.; Wright, E. R.

The physiological role of alpha-synuclein (alpha-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, alpha-syn will self-assemble into Beta-sheet rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient derived brain tissues have concluded that these inclusions are associated with Parkinson\'s disease, the second most common neurodegenerative disorder, and other synuclein related diseases called synucleinopathies. In addition, repetitions of and specific mutations to the SNCA gene, the gene that encodes alpha-syn, results in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of alpha-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 Angstrom. Here, we provide a protocol for alpha-syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step by step protocol for high resolution cryo-EM structure determination of alpha-syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high resolution cryo-EM map. This workflow resulted in a 2.04 Angstrom structure of alpha-syn fibrils with excellent resolution of residues 36 to 97 and an additional island of density for residues 15 to 22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of alpha-synuclein and other amyloid fibrils.