AbstractOver the last 20 years cryo transmission electron microscopy (cryo-TEM) has become established as a vital tool used to answer important biological questions structurally and facilitate developments in the fields of medicine and bionanotechnology. In this thesis I discuss two projects in which cryo-TEM has played a pivotal role in our understanding of complex macromolecular systems.
Type II chaperonins (known as thermosomes in archaea) are cage-like structures formed of back to back rings of subunits which close in an ATP dependent manner, sequestering unfolded proteins from the cytosol, allowing them to fold. The ATP binding and hydrolysis behaviour of the thermosome from T. acidophilum, along with a panel of mutants, had been previously characterised. Single particle electron microscopy reported in this thesis reveals the structures of the wild type, and a mutant in which the ATP hydrolysis has been blocked in one of the two subunits. The addition of ATP and aluminium fluoride captured the systems in a post-hydrolysis state and allowed comparison of the two systems. Surprisingly, the mutant was found to be in an open conformation, while the wild type was in a closed conformation similar to a previously reported crystal structure, suggesting strong cooperative behaviour between subunits in a ring. We also observed a concerted motion in all three domains of the subunits during closing, analogous to that seen for the homo-oligomeric thermosome from Methanococcus Maripaludis.
The second project reported in this thesis is a structural characterisation of a peptide-based nanoparticle, designed for applications such as drug delivery. Previous studies suggested that the modular system self-assembles into a unilamellar spherical particle. I undertook a broad ranging characterisation study which combined cryo-TEM, cryo-correlative light-electron microscopy, electron energy loss spectroscopy and small angle x-ray scattering. These revealed that the particles had a more complex, less ordered structure than previously envisaged. Cryo-TEM demonstrated internal structure which can only be attributed to component peptide. Alternative structures are discussed, and I propose it is most likely a sponge-like structure in which the self-assembling lattice propagates in three dimensions rather than two.
|Date of Award||23 Jan 2020|
|Supervisor||Paul Verkade (Supervisor) & Danielle M Paul (Supervisor)|
Structural characterisation of large macromolecular complexes using cryo-TEM
Coombs, J. (Author). 23 Jan 2020
Student thesis: Doctoral Thesis › Doctor of Philosophy (PhD)