High-resolution Cryo-EM Structure Determination of a-Synuclein—A Prototypical Amyloid Fibril
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-assemb...
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Bio-protocol LLC
2025-02-01
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| author | Juan Sanchez Joshua Pierson Collin Borcik Chad Rienstra Elizabeth Wright |
| author_facet | Juan Sanchez Joshua Pierson Collin Borcik Chad Rienstra Elizabeth Wright |
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| description | 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. |
| format | Article |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-02-01 |
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| spelling | doaj-art-8f7289c8fc9548f0886c733e92964b9b2025-02-07T08:16:46ZengBio-protocol LLCBio-Protocol2331-83252025-02-0115310.21769/BioProtoc.5171High-resolution Cryo-EM Structure Determination of a-Synuclein—A Prototypical Amyloid FibrilJuan Sanchez0Joshua Pierson1Collin Borcik2Chad Rienstra3Elizabeth Wright4Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USABiophysics Graduate Program, University of Wisconsin-Madison, Madison, WI, USA, Biotechnology Training Program, University of Wisconsin-Madison, Madison, WI, USADepartment of Biochemistry, University of Wisconsin-Madison, Madison, WI, USADepartment of Biochemistry, University of Wisconsin-Madison, Madison, WI, USANational Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, USADepartment of Biochemistry, University of Wisconsin-Madison, Madison, WI, USABiophysics Graduate Program, University of Wisconsin-Madison, Madison, WI, USA, Biotechnology Training Program, University of Wisconsin-Madison, Madison, WI, USA, National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, USA, Morgridge Institute for Research, UW-Madison, Madison, WI, USADepartment of Biochemistry, University of Wisconsin-Madison, Madison, WI, USABiophysics Graduate Program, University of Wisconsin-Madison, Madison, WI, USA, Biotechnology Training Program, University of Wisconsin-Madison, Madison, WI, USA, Cryo-Electron Microscopy Research Center, UW-Madison, Madison, WI, USA, Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, USA, Morgridge Institute for Research, UW-Madison, Madison, WI, USAThe 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.https://bio-protocol.org/en/bpdetail?id=5171&type=0 |
| spellingShingle | Juan Sanchez Joshua Pierson Collin Borcik Chad Rienstra Elizabeth Wright High-resolution Cryo-EM Structure Determination of a-Synuclein—A Prototypical Amyloid Fibril Bio-Protocol |
| title | High-resolution Cryo-EM Structure Determination of a-Synuclein—A Prototypical Amyloid Fibril |
| title_full | High-resolution Cryo-EM Structure Determination of a-Synuclein—A Prototypical Amyloid Fibril |
| title_fullStr | High-resolution Cryo-EM Structure Determination of a-Synuclein—A Prototypical Amyloid Fibril |
| title_full_unstemmed | High-resolution Cryo-EM Structure Determination of a-Synuclein—A Prototypical Amyloid Fibril |
| title_short | High-resolution Cryo-EM Structure Determination of a-Synuclein—A Prototypical Amyloid Fibril |
| title_sort | high resolution cryo em structure determination of a synuclein a prototypical amyloid fibril |
| url | https://bio-protocol.org/en/bpdetail?id=5171&type=0 |
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