Taking inspiration from nature to design new immunotherapies. Researchers aim to identify the signals that activate our immune system and distil these down to their fundamental components. This will produce new and simplified ways to stimulate our immune system and give doctors the ability to direct our body to attack cells it would ordinarily ignore, such as invading pathogens and cancer cells, and deliver powerful new therapies to tackle diseases.

Conquering the coronavirus replication centre Coronaviruses use infected cells to make copies of themselves that propagate infection. To replicate their genomes, coronaviruses hijack cellular membranes to build up specialized replication compartments. These work as control centres to make the process efficient and hide it from antiviral cellular defences. Recently, we discovered a unique protein complex that provides a gate in the membranes of these replication compartments. Here, we plan to decipher how this gate and the replication organelles are built and function, which will be key to devise plans to assault these viral fortresses to block virus replication and disease.

Recent developments in cryo-electron microscopy (cryoEM) enable the structures of individual proteins to be solved to atomic resolution and can also be used to image individual proteins within their native cellular environment, leading to 3D atlases of protein locations and information of the “molecular sociology” of cells. However, these techniques rely on imaging abundant/common proteins. Locating rare individual molecules or transient biomolecular events remains beyond the ability of cellular cryoEM. Light microscopy can be used to locate these rare proteins and events using endogenously-expressed auto-fluorescent proteins such as GFP, although the resolution of LM is not sufficient to accurately-correlate these data with cryoEM images at the single-molecule level. Super-resolution LM can, however, localise molecules within ~30-nm area, which is sufficient to identify individual proteins by cryoEM. We have recently described a technique, called super-cryoCLEM, that allows super-resolution LM to be performed at cryogenic temperatures that can be directly correlated with cryoEM. This shows promise for locating specific intracellular proteins, but requires development to become a mainstream, generally-applicable and accessible technique for cellular studies. This proposal aims to: 1) Establish methodology to perform correlated super-resolution cryoLM and cryoEM to yield a straightforward method to perform super-cryoCLEM. 2) Implement instrumentation for multicolour and 3D-resolved super-cryoCLEM. 3) Apply these techniques to address fundamental processes in immunology – how antibodies activate the innate-immune complement system on cells. We will demonstrate the potential of super-cryoCLEM by investigating immune system activation on invasive pathogens, which will increase our understanding of bacterial meningitis. We will also address complement-based anti-cancer immunotherapies by performing super-cryoCLEM on cancerous B-cells undergoing complement-dependent cytolysis. By concatenating super-cryoCLEM with structural biology, we will demonstrate the first instance of targeted structural immunology, bridging the gap between nanoscopic structural biology and macroscopic immune defence.