Asymmetric Genome Organization in an RNA Virus Revealed via Graph-Theoretical Analysis of Tomographic Data

Cryo-electron microscopy permits 3-D structures of viral pathogens to be determined in remarkable detail. In particular, the protein containers encapsulating viral genomes have been determined to high resolution using symmetry averaging techniques that exploit the icosahedral architecture seen in many viruses. By contrast, structure determination of asymmetric components remains a challenge, and novel analysis methods are required to reveal such features and characterize their functional roles during infection. Motivated by the important, cooperative roles of viral genomes in the assembly of single-stranded RNA viruses, we have developed a new analysis method that reveals the asymmetric structural organization of viral genomes in proximity to the capsid in such viruses. The method uses geometric constraints on genome organization, formulated based on knowledge of icosahedrally-averaged reconstructions and the roles of the RNA-capsid protein contacts, to analyse cryo-electron tomographic data. We apply this method to the low-resolution tomographic data of a model virus and infer the unique asymmetric organization of its genome in contact with the protein shell of the capsid. This opens unprecedented opportunities to analyse viral genomes, revealing conserved structural features and mechanisms that can be targeted in antiviral drug desig

Electron microscopy of macromolecular complexes, and their interactions with membranes

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