Using cryo-electron microscopy and molecular dynamics simulations to understand complex molecular systems

Thesis

Structural studies of biological complexes require an integration of techniques to understand both structure and function. While methods such as cryogenic electron microscopy (cryo-EM) can generate high-resolution static structures, the connection to function requires an approach which captures data over time. In this thesis I utilised molecular dynamics (MD) simulations and cryo-EM to study the structure and function of biological complexes.

MD simulations can probe the effects of mutations on the interactions between biological molecules. I investigated the consequences of changing the electrostatic surface of a potential protein nanopore on the translocation of double-stranded RNA through the pore lumen. I showed that the impact on translocation speed is due to direct interactions between the RNA and the protein, and to the indirect action of the change in ion flux.

The ribosome of the marine-dwelling archaeon Nitrosopumilus maritimus was studied using cryo-EM. I solved novel high-resolution structures of the large ribosomal subunit and built atomic models of the ribosomal RNA and proteins. I compared the presence of archaea-eukarya-specific proteins to the published structures of other archaeal ribosomes and found additional structural roles for some proteins compared to their homologues.

Many prokaryotic cells have proteinaceous lattices on their cell surface known as an S-layer. These S-layers are known to be very divergent in sequence and structure across domains down to species in the same genus. I used cryogenic electron tomography to solve medium-resolution structures of S-layers from four archaeal species, N. maritimus, Saccharolobus solfataricus, Pyrobaculum oguniense, and Pyrobaculum arsenaticum. I observed variation in S-layer architecture despite the broad presence of immunoglobulin-like folds. I used MD simulations to identify the divalent cation species likely to occupy the coordination sites I located in the Haloferax volcanii S-layer. This combined published cryo-EM data with MD data for an integrative approach to structural biology.

Authors: Ford, Z

• DOI: 10.5287/ora-pmygyoye9
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: cryo electron microscopy; cryo electron tomography; integrative structural biology; molecular dynamics simulations; protein nanopores; archaeal surface layers; archaeal ribosomes
• Subjects: Structural biology; Biochemistry