Diamond Annual Review 2020/21

112 113 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 0 / 2 1 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 0 / 2 1 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. These facilities and partnerships bring together expertise from UK universities, research institutes and industry to help us tackle 21 st century challenges through internationally leading cooperation. Integrated Facilities The Membrane Protein Laboratory (MPL) The Membrane Protein Laboratory (MPL) is a well-established, state-of- the-art facility that enables membrane protein research. Since its inception, the MPL has supported visiting researchers from around the world to work towards the visualisation of their membrane protein of interest at resolutions that allow structure-function relationships to be understood. 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 will help us to develop future therapeutics to tackle disease. The MPL is one of Diamond’s integrated facilities and located within the Research Complex at Harwell (RCaH), it provides support to scientists interested in membrane protein structural biology. Having a dedicated laboratory with cutting-edge equipment, and close to the experimental end stations and electron microscopes, greatly enhances scientists’ ability to successfully characterise these important targets. Recently, a post-doctoral researcher has been recruited to enhance the MPL’s single-particle cryo- electron microscopy (cryo-EM) capabilities, working with Diamond’s Electron Bio-Imaging Centre (eBIC) and the MPL. The MPL is open to user applications from anywhere in the world, and proteins crystallised here have been used in experiments in other facilities. Recently publishedwork in NatureCommunications 1 details research supported by MPL facilities that has led to a series of Archarhosdopsin-3 (AR3) structures. AR3 is a photoreceptor that harvests energy from sunlight to pump protons out of the cell driving ATP synthesis. Properties of AR3 make it ideally suited for optogenetic experiments, in which it is used to silence individual neurons and to detect changes in cell membrane voltage. These new structures open the way for the development of new tools and methodologies in the fields of neuroscience, cell biology and beyond. In other work, a combination of in vitro and in cellulo approaches were used to characterise disulphide bonding found in the major outer membrane protein (MOMP), an important Chlamydia vaccine target 2 . A low-resolution (~4 Å) X-ray crystal structure was used to identify a series of cysteine rich pockets and pint mutants made to investigate disulphide bonding. Working with the Central laser Facility (CLF), super-resolution fluorescence microscopy methods were used to follow localisation of MOMP in E. coli cells and measure the clustering behaviour of the different cysteinemutants. Disulphide bonding was found not to be disrupted by single point mutations and found to be compensated by neighbouring cysteines within the cysteine-dense pockets. References: 1. Bada Juarez J. F. et al. Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization. Nat. Commun. 12 , 629 (2021). DOI: 10.1038/s41467-020- 20596-0 2. Danson A. E. et al. Super-resolution fluorescence microscopy reveals clustering behaviour of chlamydia pneumoniae’s major outer membrane protein. Biology 9 , 1–16 (2020). DOI: 10.3390/biology9100344 XChem It has been a busy year for Diamond’s XChem facility for fragment screening, staying open throughout lockdown for experiments aimed at accelerating development of new treatments for COVID-19. Early in the pandemic, XChem joined a collaboration to identify fragment compounds bound to the Main protease (Mpro), a key enzyme in the life cycle of the SARS- CoV-2 virus. Since such compounds provide templates for designing bespoke molecules to block the enzyme and thus the virus, the data were placed online immediately. This triggered an avalanche of interest, and XChem became a founding partner of the COVID Moonshot, a global non-profit initiative aiming to develop a novel antiviral drug with no IP constraints, by crowdsourcing designs of new inhibitors from chemists world-wide who could mine the rich fragment data measured at Diamond. The project continues to release its data in real-time, which has driven very rapid progress. Along with collaborators, the XChem team performed screens against a further seven COVID-19 proteins, data that can trigger equally productive drug discovery efforts. By April 2021, the combined international efforts had discovered 234 fragment compounds that directly bind to sites of interest on the surface of this cohort of seven proteins, mapping out chemical motifs and protein-compound interactions. Many of these data are already public, providing large numbers of starting points for designing compounds aimed at directly-acting antivirals with novel modes of action, targeting either pandemic or else helping for pandemic preparedness. Highlighted publications: • Douangamath A. et al. Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease. Nat. Commun. 11 , 5047 (2020). DOI: 10.1038/s41467-020-18709-w • Chodera J. et al. Crowdsourcing drug discovery for pandemics. Nat. Chem. 12 , 581 (2020). DOI: 10.1038/s41557-020-0496-2 • Schuller M. et al. Fragment binding to the Nsp3 macrodomain of SARS- CoV-2 identified through crystallographic screening and computational docking. Sci. Adv. 7 , eabf8711 (2021). DOI: 10.1126/sciadv.abf8711 • Newman J. A. et al. Structure, Mechanism and Crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase. bioRxiv 2021.03.15.435326 (2021). DOI: 10.1101/2021.03.15.435326 XFEL Hub The XFEL (X-ray Free-Electron Laser) Hub at Diamond continues to provide expertise and support to the UK community engaged in serial crystallography and XFEL-related life science research; from experimental 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. Nearly all XFEL beamtime awards over the past year have been impacted by the COVID-19 pandemic. Since February 2020, our XFEL activities included more than a dozen beamtime awards fielded at XFELs SACLA (Japan), PAL- XFEL (South Korea), LCLS (USA), and the European XFEL (Germany). Nearly all have focused on time-resolved Serial Femtosecond Crystallography (SFX) data collection that was often correlated with X-ray Emission Spectroscopy (XES). Starting in August 2020 the Hub participated in all XFEL beamtime through remote connections, including the Hub-led COVID-19 experiments at LCLS and the European XFEL that probed the structure, function and dynamics of the two viral cysteine proteases: the main protease(M pro )and the papain-like protease (PL pro ). The XFEL Hub and scientists from the University of Oxford are collaborators in a newly awarded Scientific Campaign at the LCLS titled,‘Structural Dynamics of Metalloenzymes’andwill receivemultiple beamtimes over several years.The goals include “to conduct combined time-resolved X-ray crystallography and X-ray spectroscopy [on methane monooxygenase, several hydrogenases, and nitrogenases], under turn-over conditions, to elucidate details of the structural and redox changes happening at the active site of each of these systems.” The XFEL Hub initiatedmajor projects at Diamond to establish strategies for time-resolved Macromolecular Crystallography (MX) studies with on-demand sample delivery and reaction initiation methods that can be correlated with X-ray Emission Spectroscopy (XES) too. This includes establishing deeper collaborations with several XFEL facilities, including SwissFEL which may also host the capabilities developed at Diamond. Dr Allen Orville led the life science team for the UK XFEL project that was tasked by the Science and Technology Facilities Council (STFC) to create an updated science case for a UK-based X-ray Free Electron Laser (UK-XFEL). The Revised Science Case was completed and submitted to STFC/UKRI and can be downloaded​from the web link below. The project continues to seek input to the process from across the scientific community. More information can be found here: www.clf.stfc.ac.uk/Pages/UK-XFEL-science-case Highlighted publications: • Orville A. M. Recent results in time resolved serial femtosecond crystallography at XFELs. Curr. Opin. Struct. Biol. 65 , 193–208 (2020). DOI: 10.1016/j.sbi.2020.08.011 • Srinivas V. et al. High-Resolution XFEL structure of the soluble methane monooxygenase hydroxylase complex with its regulatory component at ambient temperature in two oxidation states. J. Am. Chem. Soc. 142 , 14249–14266 (2020). DOI: 10.1021/jacs.0c05613 • Ibrahim M. et al. Untangling the sequence of events during the S2 → S3 transition in photosystem II and implications for the water oxidation mechanism. Proc. Natl. Acad. Sci. U. S. A. 117 , 12624– 12635 (2020). DOI: 10.1073/pnas.2000529117 • Sethe Burgie E. et al. Photoreversible interconversion of a phytochrome photosensory module in the crystalline state. Proc. Natl. Acad. Sci. U. S. A. 117 , 300–307 (2020). DOI: 10.1073/ pnas.1912041116 Collaborations The Rosalind Franklin Institute Along with ten universities and UKRI-STFC, Diamond is a founding member of the Rosalind Franklin Institute (The Franklin) which is dedicated to bringing about transformative changes in life science through interdisciplinary research and technology development. Last year the Wellcome Trust awarded The Franklin, along with partners Medical Research Council Laboratory of Molecular Biology (MRC LMB) and Diamond, a £25m grant to support the development of three new electron imaging technologies that will have the capacity to revolutionise howwe see life, pushing the boundaries of imaging in life science. Diamond’s share of the grant is to fund the development of HeXI, a Hybrid electron - X-ray Instrument set to play a major role in drug discovery efforts. Collectively known as ‘Electrifying Life Science’, the electron imaging technologies will create globally unique capabilities for the UK. The overall project is led by Professor James Naismith, Director ofThe Franklin, Professor Sir David Stuart, Director of Life Sciences at Diamond and Joint Head of Structural Biology at the University of Oxford, Dr Gwyndaf Evans, Deputy Director Life Sciences at Diamond and Head of Technology at The Franklin and Dr Richard Henderson, group leader at MRC LMB. Together, the team will change by a factor of ten the accessibility and capability of cryo-EM, in both tomography and single particle sub-fields. HeXI will be a dedicated electron diffraction instrument embedded within Diamond’s VMXm micro/nano focus X-ray beamline facility and accessible through the Diamond user programme. It will combine and build upon state- of-the-art technologies from electron and X-ray fields to create brand new scientific opportunities for structural biology and drug discovery. HeXI will make electron diffraction easily accessible to all of Diamond’s existing life science users, as well as attract new users, to routinely study pharmaceutical compounds and their binding. The Franklin is leading on the Amplus project with Thermofisher Scientific and Diamond as collaborating partners. The project will develop instruments that will be built at The Franklin Hub and which will deliver a revolution in cryo-electron tomography – using electron microscopy to build up three- dimensional models inside the cell. This will bring into view the possibility of imaging whole cells at the atomic scale quickly and accurately. Although technically challenging, the applications are vast and include genetic disease, intracellular pathogens, understanding the mechanisms of microbial drug resistance, and observing viral infection. To make the process robust and accessible, automation will be built into every step, from sample handling to data processing. Representation of the chemicals binding to the main protease of the SARS-CoV-2 virus.