{"doi":"10.21769/bioprotoc.5171","title":"High-resolution Cryo-EM Structure Determination of a-Synuclein—A Prototypical Amyloid Fibril","abstract":"The physiological role of a-synuclein (a-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, a-syn will self-assemble into β-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 specific mutations to the SNCA gene, the gene that encodes a-syn, result 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 a-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 Å. Here, we provide a protocol for a-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 a-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 Å structure of a-syn fibrils with excellent resolution of residues 36-97 and an additional island of density for residues 15-22 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 a-syn and other amyloid fibrils. Key features • In vitro fibril amplification method yielding twisting fibrils that span several micrometers in length and are suitable for cryo-EM structure determination. • High-throughput cryo-EM data collection of neurodegenerative fibrils, such as alpha-synuclein. • Use of RELION implementations of helical reconstruction algorithms to generate high-resolution 3D structures of a-synuclein fibrils. • Brief demonstration of the use of ChimeraX, Coot, and Phenix for molecular model building and refinement.","journal":"BIO-PROTOCOL","year":2024,"id":446161,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":7,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9561,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1209133,"name":"J. Pierson","orcid":null,"position":1,"is_corresponding":false},{"id":428455,"name":"Collin G. Borcik","orcid":"0000-0002-4733-7773","position":2,"is_corresponding":false},{"id":526696,"name":"Chad M. Rienstra","orcid":"0000-0002-9912-5596","position":3,"is_corresponding":false},{"id":420653,"name":"Elizabeth Wright","orcid":"0000-0003-0712-9475","position":4,"is_corresponding":false},{"id":482370,"name":"Juan C Sanchez","orcid":"0000-0002-1178-183X","position":0,"is_corresponding":true}],"reference_count":55,"raw_metadata":null,"created_at":"2026-07-19T02:01:50.465308Z","pmid":"39959285","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}