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Cryo-electron Microscopy to Analyze the Structure of Bacterial Amyloids In Vitro
Antoine Cossa1,2, Sylvain Trépout3,4
1Laboratoire Léon Brillouin LLB, CEA, CNRS UMR12, Université Paris-Saclay, CEA Saclay, Gif-sur-Yvette, France.
Abstract:
Amyloid fibrils are aggregates of proteins or peptides. In humans, they are associated with various pathologies ranging from neurodegenerative diseases such as Alzheimer's and Parkinson's to systemic diseases like type 2 diabetes. In bacteria, amyloids can exert functional roles such as biofilm formation or gene regulation. Up to now, the aggregation mechanism leading to amyloid fibril formation is poorly understood as proteins with different amino acid sequences can fold into similar 3D structures. Understanding the formation of amyloid fibrils constitutes a central challenge for fighting major human health issues such as neurodegenerative diseases and biofilm formation in ports (implantable chambers). Since the dogma linking protein sequence, 3D structure, and function is increasingly disrupted by the growing understanding of the importance of disordered domains in proteins, it is crucial to possess a method capable of building accurate atomic models of amyloids. Aided by the leap forward of cryo-electron microscopy (cryo-EM), which can now routinely achieve sub-nanometric resolutions, it has become the method of choice for studying amyloids. In this chapter, we use the Hfq protein from Escherichia coli as an example to present general protocols in cryo-EM to unveil the structure of bacterial amyloids and improve our knowledge of their aggregation mechanism.
Insights
Amyloid fibrils, linked to human diseases and bacterial functions, are challenging to study due to complex aggregation mechanisms. Cryo-electron microscopy (cryo-EM) offers a powerful method to visualize these protein structures and understand their formation.
Area of Science:
- Structural Biology
- Biochemistry
- Microbiology
Background:
- Amyloid fibrils are protein aggregates implicated in human diseases (e.g., Alzheimer's, Parkinson's, type 2 diabetes) and bacterial functions (e.g., biofilm formation).
- The aggregation mechanism of amyloid fibrils is poorly understood, complicated by the ability of diverse protein sequences to form similar 3D structures.
- Understanding amyloid formation is critical for addressing neurodegenerative diseases and bacterial biofilm-related issues.
Purpose of the Study:
- To present general protocols using cryo-electron microscopy (cryo-EM) for determining the structure of bacterial amyloids.
- To enhance the understanding of amyloid aggregation mechanisms using the Hfq protein from Escherichia coli as a model.
Main Methods:
- Utilizing cryo-electron microscopy (cryo-EM) for high-resolution structural analysis of amyloid fibrils.
- Applying established cryo-EM protocols to bacterial amyloid structures.
Main Results:
- Demonstrated the utility of cryo-EM in resolving atomic models of amyloids.
- Provided insights into the structural basis of bacterial amyloid formation using Hfq protein.
Conclusions:
- Cryo-EM is the method of choice for studying amyloid structures due to advancements in resolution.
- Accurate atomic models of amyloids are crucial for understanding their aggregation and combating associated diseases and biofilm formation.
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01:28Cryo-electron Microscopy
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