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Abstract: Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.

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Abstract: Rabies virus can cause fatal neurological disease when access to medication is scarce. Tackling this issue will require novel vaccines and therapies to be developed and the viral surface protein may be a suitable target for these interventions. This protein arranges in triplets, but the structure of this arrangement has not yet been determined and the interaction between this triplet and therapeutic agents remains to be characterised. In a recent publication in Cell Host & Microbe, a research team at the University of Oxford collaborated with the electron Bio-Imaging Centre (eBIC) at Diamond Light Source to solve the cryo-electron microscopy (cryo-EM) structure of the triplet in complex with two therapeutic agents. Building on this work, the scientists investigated the mechanism by which licensed therapies inhibit the viral protein. The structural insight gained from this work will allow for strategic development of new vaccines and therapies against the virus.

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Abstract: Aedes aegypti has evolved to become an efficient vector for arboviruses but the mechanisms of host-pathogen tolerance are unknown. Immunoreceptor Toll and its ligand Spaetzle have undergone duplication which may allow neofunctionalization and adaptation. Here we present cryo-EM structures and biophysical characterisation of low affinity Toll5A complexes that display transient but specific interactions with Spaetzle1C, forming asymmetric complexes, with only one ligand clearly resolved. Loop structures of Spaetzle1C and Toll5A intercalate, temporarily bridging the receptor C-termini to promote signalling. By contrast unbound receptors form head-to-head homodimers that keep the juxtamembrane regions far apart in an inactive conformation. Interestingly the transcriptional signature of Spaetzle1C differs from other Spaetzle cytokines and controls genes involved in innate immunity, metabolism and tissue regeneration. Taken together our results explain how upregulation of Spaetzle1C in the midgut and Toll5A in the salivary gland shape the concomitant immune response.

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Abstract: Neurodegenerative disorders are characterised by the deposition of proteinaceous aggregates in the brain. In the case of synucleinopathies, such as Parkinson’s disease, the aggregates are mainly composed of α-synuclein (αSyn). The aggregation process of αSyn is toxic for neurons but the cause of the toxicity remains elusive. In vitro, the constitutively expressed cytosolic heat shock 70 protein, Hsc70, together with two cochaperones, the J-domain protein DNAJB1 and the nucleotide exchange factor Apg2, can disassemble αSyn amyloid fibres and small oligomers. However, the model is incomplete, lacking structural as well as mechanistic details. To address these two issues (αSyn aggregation in cells and the incomplete model of αSyn disaggregation in vitro), I have used electron and fluorescence microscopy and biochemical assays. Correlating fluorescence images and cryo tomograms, I have investigated the structure of αSyn aggregates in a yeast model. αSyn colocalises at the plasma membrane and forms cytosolic inclusions rich in vesicles and lipid droplets. Using cryo electron microscopy (cryo-EM) and tomography, I have shown that recombinant αSyn does not form large oligomers on the surface of tubulated liposomes. Using single particle cryo-EM, I have studied the complex formed of αSyn amyloid fibres and a mutant of DNAJB1 lacking the J-domain and G/F linker (ΔJ-DNAJB1). A low-resolution reconstruction of the complex was generated in which horseshoe-like densities, identified as ΔJ-DNAJB1, surrounded the fibre. I also produced a high-resolution map of the αSyn fibre showing a new conformation. Lastly, using cryo-EM, fluorescence microscopy and biochemical assays, I have investigated the function of Apg2 in disaggregation. Unexpectedly, Apg2 enhances Hsc70 recruitment to the fibres, preferentially at one end. This Apg2-induced recruitment regulates the disaggregation activity. The work in this thesis provides new information about αSyn aggregation, its toxicity and its disaggregation by the Hsc70 system.

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Abstract: In oxygenic photosynthesis, the cytochrome b6f (cytb6f) complex links the linear electron transfer (LET) reactions occurring at photosystems I and II and generates a transmembrane proton gradient via the Q-cycle. In addition to this central role in LET, cytb6f also participates in a range of processes including cyclic electron transfer (CET), state transitions and photosynthetic control. Many of the regulatory roles of cytb6f are facilitated by auxiliary proteins that differ depending upon the species, yet because of their weak and transient nature the structural details of these interactions remain unknown. An apparent key player in the regulatory balance between LET and CET in cyanobacteria is PetP, a ~10 kDa protein that is also found in red algae but not in green algae and plants. Here, we used cryogenic electron microscopy to determine the structure of the Synechocystis sp. PCC 6803 cytb6f complex in the presence and absence of PetP. Our structures show that PetP interacts with the cytoplasmic side of cytb6f, displacing the C-terminus of the PetG subunit and shielding the C-terminus of cytochrome b6, which binds the heme cn molecule that is suggested to mediate CET. The structures also highlight key differences in the mode of plastoquinone binding between cyanobacterial and plant cytb6f complexes, which we suggest may reflect the unique combination of photosynthetic and respiratory electron transfer in cyanobacterial thylakoid membranes. The structure of cytb6f from a model cyanobacterial species amenable to genetic engineering will enhance future site-directed mutagenesis studies of structure-function relationships in this crucial ET complex.