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.
Keywords: Cryo-electron microscopy; E. coli; Functional amyloid; Hfq.
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.