Over the last ~4 years, cryo-electron microscopy (EM) has undergone a ‘revolution’, thanks to advances in microscope hardware, such as direct electron detectors, and image processing algorithms. This has improved the quality of data that can be obtained using cryo-EM, and so a diverse range of biological problems can now be effectively tackled using this technique. Here, two biological systems are examined using different cryo-EM imaging modalities.
A large number of human diseases are associated with the formation of amyloid fibrils. Amyloid-membrane interactions may play a key role in amyloid mediated cytotoxicity. In Chapter Three, a combination of liposome dye release assays and cryo-EM is used to investigate the effect of amyloid fibrils on membranes of varying compositions. Solution conditions such as pH, alongside lipid composition, were found to have a profound effect on the propensity of β-2-microglobulin (β2m) amyloid fibrils to perturb membranes. In Chapter Four, subcellular fractionation and cryo-EM and tomography were used to further probe the nature of interactions between β2m amyloid fibrils and cellular membranes. Cryo-electron tomography was used to reveal 3D detail of unique interactions at molecular resolution.
In Chapter Five, structural properties of the Leviviridae family of bacteriophages, a model family of spherical viruses, were investigated using cryo-EM. The structure of Qβ bacteriophage at 4.2 Å is presented. Using this electron density map, an existing X-ray crystal structure is refined to yield a better quality model of the Qβ capsid. Asymmetric reconstructions were used to generate insight into genome organisation and capsid assembly.
This work demonstrates the utility of cryo-EM as a flexible technique to tackle a broad range of research questions by providing structural information at different resolutions.
