9CK3
Cryo-EM structure of in-vitro alpha-synuclein fibril
Summary for 9CK3
| Entry DOI | 10.2210/pdb9ck3/pdb |
| EMDB information | 45639 |
| Descriptor | Alpha-synuclein (1 entity in total) |
| Functional Keywords | alpha-synuclein, fibril, amyloid, structural protein |
| Biological source | Homo sapiens (human) |
| Total number of polymer chains | 12 |
| Total formula weight | 173713.30 |
| Authors | Sanchez, J.C.,Borcik, C.G.,Tonelli, M.,Sibert, B.,Rienstra, C.M.,Wright, E.R. (deposition date: 2024-07-08, release date: 2024-07-24, Last modification date: 2025-08-20) |
| Primary citation | Sanchez, J.C.,Pierson, J.A.,Borcik, C.G.,Rienstra, C.M.,Wright, E.R. High-resolution Cryo-EM Structure Determination of a-Synuclein-A Prototypical Amyloid Fibril. Bio Protoc, 15:e5171-e5171, 2025 Cited by PubMed 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. PubMed: 39959285DOI: 10.21769/BioProtoc.5171 PDB entries with the same primary citation |
| Experimental method | ELECTRON MICROSCOPY (2.04 Å) |
Structure validation
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