104 105 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 Integrated Facilities and Collaborations A s a world-leading centre for synchrotron science and a cornerstone of a world-class site for scientific discovery and innovation at Harwell, Diamond Light Source has powerful synergies with its neighbouring research institutes and beyond the campus, through collaborations and shared visions. The integrated facilities at Diamond present academic and industrial users with a one-stop- shop for research opportunities, enabling them to combine cutting-edge techniques and capabilities to advance their studies. Diamond participated in 20 grants in the fiscal year 2021 – 2022, and Diamond researchers show a greater interest in collaborative grants. You will find in this section some highlighted collaborations. Integrated Facilities The Membrane Protein Laboratory (MPL) The Membrane Protein Laboratory (MPL), led by Dr Andrew Quigley at Diamond Light Source, has been awarded £1.5m of Wellcome funding to support the laboratory’s research for a further five years. The MPL is one of Diamond’s integrated user facilities and is located within the Research Complex at Harwell. Its focus is understanding the structure of membrane proteins through the delivery of high-quality samples to Diamond’s beamlines and microscopes. This new funding fromWellcome, a shareholder of Diamond, will enable wider sample support to further Diamond beamlines and microscopes and form a foundation for integrative membrane protein structural biology. Membrane proteins are found at the junctions between the outside world and the inner workings of the cell. Multicellular organisms such as humans use membrane proteins for communication, to acquire nutrients and detect threats. Membrane proteins are important targets for biomedicine, with over half of all medicines altering membrane protein function. Understanding the structure and function of these proteins in isolation as well as within the wider cellular context will help us to develop new therapeutics to tackle disease. Visiting scientists to the MPL can spend anywhere between a day and a year in its labs, supported by state-of -the art equipment and its experienced support scientists. Recently published work in Nature Communications 1 details the molecular basis of regulation of bacterial capsule assembly by Wzc. Wzc is the regulator behind the formation of extracellular polysaccharides that help bacteria to evade the human immune response. The authors show that dephosphorylation of Wzc leads to the assembly of an octomer. This is the molecular switch that enableWzc to cycle through on and off states controlling the passage of lipid linked polysaccharides. In other work, MPL scientists have collaborated with researchers from Protein Production UK (PPUK) at the Rosalind Franklin Institute supporting a systematic study looking at the small-scale transient expression of Eukaryotic membrane proteins in Expi293F cells 2 . Small-scale screening allows the rapid optimisation of samples that can be used for cryo-electron microscopy and crystallisation. Detailed protocols were published in Methods in Molecular Biology . 1. Yang, Y., Liu, J., Clarke, B.R. et al. The molecular basis of regulation of bacterial capsule assembly byWzc. Nat Commun 12, 4349 (2021). https:// doi.org/10.1038/s41467-021-24652-1 2. Krasnoselska GO, Dumoux M, Gamage N et al . Transient Transfection and Expression of Eukaryotic Membrane Proteins in Expi293F Cells and Their Screening on a Small Scale: Application for Structural Studies. Methods Mol Biol. 2021;2305:105-128. doi: 10.1007/978-1-0716-1406-8_5. XChem Alongside the full return of the XChem user programme, including welcoming our users back on-site, the XChem team has continued to support the COVID Moonshot, a global open science, structure enabled drug discovery campaign targeting the SARS-CoV2main protease. By leveraging crowdsourced medicinal chemistry design, high throughput structural biology, machine learning and exascale molecular simulations, the collaboration discovered a novel chemical scaffold and developed it into orally bioavailable inhibitors with clinical potential within two years. These inhibitors are currently being evaluated in pre-clinical studies funded by an £8 million award fromWellcome on behalf of the COVID-19 Therapeutics Accelerator (https://www.diamond. ac.uk/Home/News/LatestNews/2021/27-09-21.html) . All compound designs, structural data, assay data and synthesised molecules have been shared rapidly and openly, creating a rich, intellectual property free knowledge base that can be exploited by the wider scientific community. To enable increased throughput of XChem to both meet user demand for ultra-high-throughput fragment screening and to exploit new experimental modalities, such as screening membrane proteins and producing data to drive machine learning and artificial intelligence approaches, it has been proposed as part of the Diamond-II project to build a newbeamline capable of underpinning the next revolution in rational drug discovery over the coming decade. This new beamline, K04, will be a flagship project of Diamond-II with building taking place in the run up to the Diamond dark period, minimising the downtime of a key UK infrastructure. Highlighted publications: • The COVID Moonshot Consortium. Open science discovery of oral non-covalent SARS-CoV-2 main protease inhibitor therapeutics. bioRxiv , (2021) DOI: 10.1101/2020.10.29.339317 • Singh, A. K. et al. Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - targeting P-pocket by fragment screening. Nat Commun . 8 , (2021). DOI: 10.1038/s41467-021-27409-y • Piticchio, S. G. et al . Discovery of novel BRD4 ligand scaffolds by automated navigation of the fragment chemical space. J. Med. Chem . 64 , 17887–17900 (2021). DOI: 10.1021/acs.jmedchem.1c01108 • Bajusz, D. et al. Exploring protein hotspots by optimized fragment pharmacophores, Nat Commun. 12 , 3201 (2021). DOI: 10.1038/s41467- 021-23443-y • Mahy,W. et al . 5-Phenyl-1,3,4-oxadiazol-2(3H)-ones are potent inhibitors of notum carboxylesterase activity identified by the optimization of a crystallographic fragment screening hit. J. Med. Chem. 63, 12942–12956 ( 2020). DOI: 10.1021/acs.jmedchem.0c01391 XFEL Hub The X-ray Free Electron Laser (XFEL) Hub at Diamond continues to provide expertise and support to the UK community engaged in serial crystallography and XFEL-related life science research. This ranges fromexperimental conception to beamtime proposals, through sample preparations and testing, to XFEL data collection, analysis, and publication. Our Diamond-based activities continue to include organising and running the block allocation group “Dynamic Structural Biology at Diamond and XFELs” for serial crystallography and time-resolved studies at various MX beamlines at Diamond. This fiscal year, members of the Hub participated in 13 XFEL experiments at the LCLS in the USA, PAL-XFEL in Korea, or the European XFEL in Germany, mostly by remote access and with reduced scientific scope. Two XFEL experiments at PAL-XFEL were cancelled due to the pandemic. In March 2022, several members of the XFEL Hub travelled to the LCLS to participate on-site in experiments in time-resolved Serial Femtosecond Crystallography (tr-SFX) correlated with X-ray Emission Spectroscopy (tr-XES). Highlighting the synergistic overlap and technology transfer between XFEL and synchrotron facilities, the XFEL Hub continued two major projects at Diamond that will establish methods for time-resolved serial crystallography studies using on-demand sample delivery and reaction initiation strategies that can be correlated with tr-XES too. The plans also include a deeper collaboration with one or more XFEL facilities, including SwissFEL, which may also host the sample delivery capabilities developed at Diamond. The Hub has been testing prototypes for sample delivery and XES data collection with von Hamos geometry at Diamond beamline VMXi. Orders have been placed for analyser crystals to enable XES from copper- and/or iron-dependent metalloenzymes. Commissioning activities for these two projects are anticipated later in the year and further. Highlighted publications: • Rabe, P. et al . X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis, Science Advances 7 , (2021). DOI: 10.1126/sciadv.abh0250 • Butryn, A. et al. An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography, Nature Communications 12 , 4461 (2021). DOI: 10.1038/s41467-021-24757-7 Collaborations The Rosalind Franklin Institute The Diamond-Franklin collaboration in electron imaging and diffraction methods ismoving fromstrength to strength.This past year has seen the delivery of the Franklin's first Titan Krios microscope, Dorothy , and the second-generation plasma focused ion beam (pFIB), called Franklin to enable large volume cellular tomography using novel sample geometries as part of a close collaboration with Thermo Fisher Scientific. The developments are funded by Wellcome through the Electrifying Life Science grant that also funds the development of the HeXI dedicated electron diffraction beamline. The Dorothy microscope was delivered in July 2021, commissioned by Diamond and Franklin staff throughout the MPL Laboratory in RCaH. XChem campaigns on SARS-CoV-2 targets.