High-Resolution Cryo-EM Structure Determination of α-Synuclein - A Prototypical Amyloid Fibril

The physiological role of -synuclein (-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, -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 Parkinsons 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 -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 -syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 [A]. Here, we provide a protocol for -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 -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 [A] structure of -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 -synuclein and other amyloid fibrils. Key FeaturesO_LIIn vitro fibril amplification method yielding twisting fibrils that span several micrometers in length and are suitable for cryo-EM structure determination. C_LIO_LIHigh-throughput cryo-EM data collection of neurodegenerative fibrils, such as alpha-synuclein. C_LIO_LIUse of RELION implementations of helical reconstruction algorithms to generate high-resolution 3D structures of a-synuclein fibrils. C_LIO_LIBrief demonstration of the use of ChimeraX, Coot, and Phenix for molecular model building and refinement.s C_LI Graphical overview of -synuclein fibrilization and cryo-EM structure determination-synuclein protein expression and purification is followed by a fibrilization protocol yielding twisting filaments that span several micrometers in length and are validated by negative stain transmission electron microscopy (NS-TEM). The sample is then vitrified, followed by cryo-EM data collection. Real-space helical reconstruction is performed in RELION to generate an electron potential map that is used for model building. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/613698v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1b008forg.highwire.dtl.DTLVardef@c1c1e1org.highwire.dtl.DTLVardef@9fc539org.highwire.dtl.DTLVardef@14d3918_HPS_FORMAT_FIGEXP M_FIG C_FIG