A famous biochemist, Arthur Kornberg, won the Nobel Prize for his work on the mechanisms by which DNA copies itself from cell to cell, generation to generation. But, he was acutely aware that whilst DNA (and by association, RNA) are the blueprint, the true vocation of life lies in the actions of the machines that are described in the nucleic acid blueprint, the proteins. To truly understand living processes, we need to gain a detailed quantitative understanding of the protein world. And, just as the field of genomics has transformed our knowledge of DNA, so an equivalent field of 'proteomics' has hugely advanced our understanding of the protein world. The core technology in proteomics is based on sophisticated mass spectrometers, capable of analysing one million millionths of a gram of peptide in exquisite detail (we use peptides as the proxy molecule for their parent proteins). But sophisticated as they are, mass spectrometers all have one intrinsic limitation - they give different signal intensities for different peptides from the same protein, even though they are in the same amount. Yet, to understand how a cell is the manifestation of the proteins it contains, we need to be able to measure exactly how many copies of any one protein there are. To overcome this limitation, we use accurately known standards that are co-analysed by the mass spectrometer, with the advantage that the standard and true cellular protein can be separately measured because we engineer the standard to be 'heavier' and thus discernible in the mass spectrometer. Thus, if we add 1000 molecules of a standard, and the cell component gives us a signal that is twice as large, we can confidently assert that the sample contains 2,000 copies of that protein. About 12 years ago, we invented a new method to generate large numbers of standards for quantitative proteomics. We created new 'designer proteins', never seen before on the planet, that could be made, in heavy form, by simple production in bacteria. These artificial proteins each contained peptide standards for up to 50 proteins. Because these proteins were pre-designed in terms of the proteins that were encoded within it, it meant that they were not always perfectly tuned to the needs of individual scientists. What was needed was the ability to 'build your own' designer protein. In this proposal, we have devised a way to do exactly this. In future, no matter what the system of interest, scientists will be able to 'dial up' their interesting proteins, and we will be able to assemble, 'a la carte' a protein standard. We will create a library of building blocks (we call them 'Qbricks', short for 'Quantification biobricks') and using advanced synthetic biology methods of DNA manipulation we will be able to create, in two days, the perfect standard protein for their research. We call these 'ALACATs' because of the 'à la carte' design philosophy. This is a revolutionary approach to absolute quantitative proteomics, and has huge potential to enhance our understanding of the protein world. This project will establish the core technology and methodologies, and build a set of Qbricks that will be used to create standards and research tools for the proteomics community. We will show how the ALACAT philosophy can be developed as a technical resource, readily drawn upon by many research groups, and thus, enabling a broad series of research programmes in a sustainable fashion.

We will install a 300kV aberration corrected STEM that utilises artificial intelligence (AI) to simultaneously improve the temporal resolution and precision/sensitivity of images while minimizing the deleterious effect of electron beam damage. Uniquely, this microscope goes beyond post-acquisition uses of AI, and integrates transformational advances in data analytics directly into its operating procedures - experiments will be designed by and for AI, rather than by and for a human operator's limited visual acuity and response time. This distributed algorithm approach to experimental design, is accomplished through a compressed sensing (CS) framework that allows measurements to be obtained under extremely low dose and/or dose rate conditions with vastly accelerated frame rates. Optimizing dose / speed / resolution permits diffusion to be imaged on the atomic scale, creating wide-ranging new opportunities to characterise metastable and kinetically controlled materials and processes at the forefront of innovations in energy storage and conversion, and the wide range of novel engineering/medical functionalities created by nanostructures, composites and hybrid materials. The microscope incorporates in-situ gas / liquid / heating / cryo and straining / indentation stages to study the dynamics of synthesis, function, degradation / corrosion and regeneration / recycling on their fundamental length and time scales. It will be housed in the Albert Crewe Centre (ACC), which is a University of Liverpool (UoL) shared research facility (SRF) specialising in new experimental strategies for high-resolution/operando electron microscopy in support of a wide range of academic/industrial user projects. UoL supports all operational costs for the SRFs (service contracts, staff, consumables, etc), meaning that access to the microscope will always be "free at the point of use" for all academic users. This open accessibility is managed through a user-friendly online proposal submission and independent peer review mechanism linked to an adaptable training/booking system, which allows the ACC to provide extensive research opportunities and training activities for all users. In particular, for early career scientists, we commit experimental resources supporting UoL's commitment to the Prosper project for flexible career development and the Research Inclusivity in a Sustainable Environment (RISE) initiative that is creating a research culture maximising inclusivity and diversity synergistically with encouraging creativity and innovation. This new microscope aligns to several priority areas of research into materials, energy and personalised medicine at the UoL, priority research areas of EPSRC and national facilities in electron microscopy, imaging and materials science, and UKRI plans for infrastructure growth (https://www.ukri.org/research/infrastructure/). In addition to supporting extensive research programs at UoL linked to investments in the Materials Innovation factory (MIF), the Stephenson Institute for Renewable Energy (SIRE) and the new Digital Innovation Facility (DIF), this unique and complimentary microscope will be affiliated to and leverage from partnership with the national microscopy facilities at Harwell (ePSIC) and Daresbury (UKSuperSTEM) and the Henry Royce Institute, as well as form extensive research links to the Rosalind Franklin Institute and the Faraday Institution. We have established (and will expand through outreach activities) an extensive network of partners/collaborators from the N8 university group, Johnson Matthey and NSG, the Universities of Swansea, Birmingham, Warwick, Oxford, Cambridge, Loughborough, Edinburgh and Glasgow and Northwest UK area SME's as well as from universities in the USA, Ireland, Germany, Japan, France, Italy, Denmark, India, Singapore, China, South Africa and Spain who will create a dynamic, innovative and collaborative community driving the long-term research impact of this facility.

The Life Sciences sector forms a key part of the UK economy: it employs over 220,000 people, contributes significantly to GDP and UK balance of trade, and is crucial for developing leading-edge treatments. It is underpinned by the UK's world-leading research base in the health and life sciences. Many key research breakthroughs are, in turn, enabled by advances in engineering and physical sciences (EPS) research - which provide ever more sophisticated instrumentation and methods to support the study of living organisms (from microbes to plants, animals and the human body) and biological processes (including both disease pathology and drug action). R&D across all parts of this ecosystem - from fundamental understanding to applied research to product development - is crucial for the delivery of long-term economic growth and continued advances in agriculture, food security, healthcare and public health. Historic models of innovation have often been linear, involving a degree of serendipity. Disruptive technologies and scientific breakthroughs will be accelerated if physical scientists, engineers, life scientists and industry work together, and at scale. This is the domain of the Rosalind Franklin Institute (RFI): with a focal point (Hub) at Harwell Science and Innovation Campus, linked to formal Spokes in leading HE ls across the UK, it will integrate complementary expertise from academia and industry to create a national centre of excellence for methods development at the convergence of the physical and life sciences. The diverse chemical control and editing of biological samples has profound potential well beyond current methods in traditional modes of Physical or Biological Sciences. This will ultimately complement and iteratively determine the value of other unifying goals of the Franklin such as Imaging. The ability to 'edit-characterize-and-image' could provide a grand challenge method for not only observing (imaging) biology but controlling (editing/treating) and hence understanding it in its post-translational state as a new mode of Medicine. The roadmap for Next Generation Chemistry (NGC) will unify several sub-projects that will span this spectrum of chemical control and understanding of biology. The NGC theme uses the rubric of: Construct (Synthetic Biology) -> Understand (Chemical Biology) -> Control (Chemical Medicine) In Construct, we aim to build and edit complex biological entities from the bottom-up. In Understand, we characterize and hence correlate built and modulated structures with function. In Control, we exploit this understanding to iteratively modulate and correct function with application primarily in Medicine and Biotechnology.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

The Nobel prize-winning technology of cryogenic Electron Microscopy (cryoEM) has transformed structural biology research, furthering our knowledge of biology, providing novel insights into the molecular mechanisms of health and disease, enabling drug design, and driving engineering biology efforts. Until recently, high-resolution cryoEM was limited to purified proteins and complexes, which necessitates removing the protein from its native environment. We therefore lose in situ information, which contains the functional data about the cellular context. Cryogenic electron tomography (cryoET) provides this information, but samples must be less than 200 nm thick for high-resolution imaging, whereas mammalian cells are >5000 nm thick, which completely precludes imaging. To produce thin samples, the optimal method is focussed ion beam (FIB) milling, performed at cryogenic temperatures (-180°C). Such a cryo-FIB removes material with nanometre precision, leaving behind a lamella - a thin slice - through the sample (e.g., cell, tissue, biopsy, etc). Cryo-FIB-SEMs contain integrated fluorescence modules that allow for targeted milling towards the fluorescent regions/molecules of interest, making the process more efficient and time-saving. CryoEM in the south-west of the UK is world-leading and highly collaborative, supported by the GW4 facility for high-resolution cryoEM. It is used extensively by the Universities of Bath, Bristol, Cardiff and Exeter (GW4) and beyond. Since 2017, the facility has enabled the determination of dozens of structures. However, research has so far largely focussed on single-particle analysis approaches. Driven by the aspiration and need of GW4 researchers to incorporate in situ structural biology using cryoET, the Universities of Bristol (the host institute) and Exeter have recently invested in equipment and personnel to expand the region's state-of-the-art cryoEM capabilities. In particular, the recent recruitment of Thom Sharp, a cryoET specialist, to Bristol was borne with that vision in mind. We seek to acquire the first cryo-FIB-SEM in the south-west of UK dedicated to strengthen in situ structural biology research in the region. Various types of integrated fluorescence cryo-FIB-SEMs are available; some are designed for the one single task of lamella preparation, which limits the application spectrum of such an (expensive) instrument. Here, we are applying for a microscope that, in addition to the targeted lamella preparation, will allow for "routine" cryo-SEM applications and uniquely incorporate elemental analysis capabilities (EDS) under cryo-conditions without compromising the cryo lamella preparation capabilities. Such an instrument would provide completely novel capabilities for GW4 but importantly will also replace an over 18-year old cryo-SEM and integrate the new capabilities (targeted lamella preparation and cryo-EDS) with existing ones ("routine" cryo-SEM) into 1 instrument to increase sustainability rather than running 2 individual instruments. The new instrument will support a very wide range of research projects, and herein we demonstrate the need for these new capabilities by 4 detailed case studies from GW4 researchers supplemented with the titles of an additional 18 planned projects, addressing fundamental biological understanding, advancing cell biology for an integrated understanding of health, engineering biology, and technology development, all key BBSRC strategic areas.