Structural biology has changed our understanding of life on the atomic scale. However, we still do not understand how molecules in the context of an organism carry out their functions well enough to understand how to treat complex diseases. Recent efforts to turn structural biology into a technique capable of looking at the structure of molecule within cells have led to a step change in approach and has led to an atomic scale understanding of molecules within tissues. However, for tissues, all-sample mapping under cryogenic conditions is still not possible meaning expanding the applications of structural cell biology to distinct processes in tissues is lacking. Current scanning electron microscopes (SEM) are optimised for tissue that has been fixed, resin-embedded and stained with heavy metals, where the requirement for charge mitigation and image optimisation for high resolution imaging is less challenging. In contrast, samples that have been vitrified for structural studies downstream are fully hydrated, and exhibit a high degree of image artefacts due to the remaining water content. The ability to image these samples with their native frozen-hydrated environment intact is still challenging, and requires new image acquisition methods to embed them as a routine tool for correlative multimodal imaging. As part of this proposal, we aim to optimise SEM imaging in our recently published multimodal workflow by implementing sparse and irregular optimised scanning routines to overcome the charge build up that occurs with standard imaging regimes. Our preliminary development work shows that such an approach can make a significant difference in the image quality of natively preserved cells and tissues, on an instrument which is integral to a structural biology workflow. This represents a step-change, where features of high relevance can be targeted, imaged with high fidelity and processed for subsequent analysis. Such an instrument will represent a transformative ability to image molecular processes in tissues, facilitating efforts to combat neurodegeneration, cardiomyopathies, liver disease and kidney dysfunction.

World Health Organization (WHO) has declared antimicrobial resistance as one of the top 10 global public health threats facing humanity. Thus, there is an urgent need to restock our dwindling drug pipeline to combat rising antimicrobial resistance. Natural products (NPs) have been an unparalleled source of bioactive compounds with numerous success stories, offering compounds with highly desirable qualities: exploiting novel targets and/or possessing unique chemical scaffolds. This proposal aims to deliver bioactive NPs by: 1. Deciphering NP biosynthesis: Non-ribosomal peptide synthetases (NRPSs) are megaenzymes with many moving parts and reactions centres producing a plethora of bioactive NPs. Despite being extensively studied NRPS gatekeeping function, substrate transfer and reactive intermediate chaperoning are unclear. To fill these gaps in our understanding we plan to structurally characterise NRPS in action in their native subcellular environment using cryogenic electron tomography (Cryo-ET) - a technique that to date has not been used for NRPS structural studies. This coupled with deep learning framework will allow us to visualize how structural heterogeneity effects domain-domain interactions in situ, while reactive intermediates are channelled across the assembly line with high catalytic efficiency and precision. The in-depth mechanistic understanding would catapult NRPS reengineering efforts to generate peptides with new or improved biological activities. 2. Isolation of novel NP: Myxobacteria are well-established factories of NPs, including ribosomally synthesized and post-translationally modified peptides (RiPPs). We plan to exploit as yet under-explored myxobacterial RiPP potential by integrating genome-mining tools with streamlined biosynthetic pathway refactoring and heterologous expression of RiPPs in a semi high-throughput fashion. Subsequently, the isolated NPs and the bioengineered derivatives would be evaluated for antimicrobial activity.

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Seeing life in 4-dimensions (space and chemistry) is crucial in understanding development and disease. Some of the most pressing needs for new insight in bioscience: neurodegeneration, cancer, cellular transport and metabolomics, bacterial multi-resistance, all require new tools and methods able to probe life in new ways across scale to enable new insights for treatment. To do this requires the delivery of these tools and methods in the form of next generation instrumentation for meso- and nanoscale imaging and chemical mapping of cells and tissue. Here we aim initially to demonstrate how 4D chemical and spatial imaging could be possible through the combination of scanning electron microscopy (SEM), sample milling via plasma focused ion beams (pFIB) integrated with secondary ion mass spectrometry (SIMS). This multichannel, spatial and chemical, imaging of volumes would enable mapping and navigation through complex biological samples at the tens of nanometre scale and the subsequent targeted preparation of thin lamella samples for nanometre resolution analysis by cryo-electron tomography. Advances in the field of Cryo-Electron Tomography (Cryo-ET) now enables structures in cells and tissues to be imaged on a pseudo-atomic scale within their native context. Simultaneous advances in mass spectrometry imaging (MSI) makes detection of the metabolome at subcellular resolution and under cryo-conditions possible. Furthermore, current research shows that proteins, peptides, lipids and metabolites can all be identified and localised within a single sample for a truly multi-omic approach. However, bridging the gap in size domains and getting structural and chemical information on the same areas of interest is a significant challenge. In this project we aim to explore how these new technologies could be used to bridge this gap and radically enhance each other through a series of proof-of-concept experiments, on cell and tissue samples, that will enable design of transformative technology in life sciences. Work at the Rosalind Franklin Institute is driven by a clear aim of making transformative leaps through development of new instrumentation, creating new ways of working and integrating previously disparate approaches. This conceptual design would deliver a completely new type of instrument, capable of exploring biological tissues in a way previously not possible - bringing together high-resolution SEM imaging with SIMS imaging to create a 4D FIB-SEM-SIMS system that overlays chemical information, eventually in the form of protein identification, onto the high-resolution 3D spatial map obtained with SEM.

Harwell Open Week will take place from 2nd to 6th July 2024, with an anticipated 10,000 members of the public expected to visit the site over the full event, of which approximately 2,000 will attend activities as part of two school open days. Historically, the school open days have been oversubscribed, meaning some schools are turned away due to space and logistics constraints, and in some cases, schools have been unable to attend due to financial constraints. This project will address the unmet needs of primary schools unable to attend Harwell Open Week and deepen connections with those who do attend. To do this, we will supply every class with a tried and tested educational resource built around a STFC-enabled citizen science project ("Virus Factory in Schools"), host online Q&As between schools and researchers, and provide those schools who could not attend the open week with alternative in-person engagement at Harwell Campus. In doing so, this project will widen STFC reach beyond the capacity of the main campus event, highlight STFC-enabled and people-powered research, and give all students direct contact with researchers working at Harwell Campus.