Diamond Annual Review 2019/20

92 93 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 1 9 / 2 0 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 1 9 / 2 0 Integrated Facilities and Collaborations W ith a large number of scientific collaborations and integrated facilities on site, Diamond Light Source is in the unique position of being able to offer a diverse and powerful resource for the advancement of research. With a breadth of techniques available, and a dedicated team of expert scientists, these complementary assets continue to allow us to enable inter- and multi-disciplinary research. The integrated facilities at Diamond have gone from strength to strength, allowing more in-depth and longer-term research to take place, truly taking advantage of the expertise here on campus.With exciting developments in our state-of-the-art facilities, we are able to provide new capabilities through our complementary facilities. Our collaborations continue to expand and improve, strengthening and gaining momentum as we progressed through 2019. Through the evolution of our existing partnerships, our collaborations have achievedworld-changing projects this year acrossmany disciplines. One such collaborationwas announced between Diamond and The Pirbright Institute, cementing a long and productive association that has resulted in vital research developments. These uniquely placed and complimentary resources promise to continue to address 21st century challenges through internationally- leading cooperation. Integrated Facilities The electron Bio-Imaging Centre (eBIC) eBIC is the first high-end cryo-electron microscopy (cryo-EM) facility worldwide to be embedded in a synchrotron, and its user operations mirror the well-established synchrotron beamline model. eBIC was established following the initial award of a £15.6 million grant from the Wellcome Trust, the Medical Research Council (MRC), and the Biotechnology and Biological Sciences Research Council (BBSRC). After successful review by the Scientific Advisory Committee (SAC) in 2018, eBIC is now fully integrated into Diamond's core programme as part of the Biological Cryo-Imaging science group. The partnerships, and the unique location of eBIC at Diamond, enable scientists to combine their techniques withmany of the other cutting-edge approaches that the synchrotron offers. eBIC provides scientists with state-of-the-art experimental equipment and expertise in the field of cryo-EM, for both single particle analysis and cryo- electron tomography. For the academic user programme, eBIC houses four Titan Krios microscopes, a Talos Arctica, a Scios and an Aquilos cryo-FIB/SEM. In addition, a partnership with the University of Oxford allows users to access a Krios in high containment located at Oxford, and a collaboration with Thermo Fisher Scientific provides anotherTitan Krios and a 200 keVmicroscope (Glacios) dedicated for industry users. New cryoEM capabilities have been developed and/or incorporated in eBIC, these include: • All 4 Krios are equipped with K3 detector • High-throughput data collection with up to 800 movies/hour was enabled with aberration-free image shift (AFIS) and soon with fringe-free imaging (FFI) • Development of a cryoCLEM workflow with recent acquisition of Leica cryoCLEM • EstablishmentofaRelionpipelinewith ISPyBforon-the-flySPAprocessing, while in-house tomography pipeline showed promise There have been several key developments and events for eBIC, including a new collaboration with The Rosalind Franklin Institute and Thermo Fisher Scientific on tomography. eBIC also held a number of successful workshops over the last year, including the 4th ‘Cryo-EM Sample Preparation Workshop’ in November 2019, which focused on teaching new cryo-EM users how to prepare samples for imaging in a three day, intensive hands-on course. This was supported by the electron microscopy facility at the Astbury Biostructure laboratory at the University of Leeds, the Institute of Structural and Molecular BiologyatBirkbeckCollege,theDivisionofStructuralBiologyattheUniversityof Oxford (STRUBI), MRC Laboratory of Molecular Biology,Thermo Fisher Scientific, Leica Biosystems, TTP Labtech, CryoSol-World and Quorum Technologies. The first hands-on practical microED workshop was held in November 2019 in collaboration with Thermo Fisher Scientific, which attracted overwhelming applications. Leading experts in microED, includingTamir Gonen, Xiaodong Zou and DavidWaterman, presented lectures in the three day workshop. The Scios and Aquilos cryo-focused ion beam scanning electron microscope at eBIC started its user program inMay 2019, following a commissioning period (October 2018 – March 2019). eBIC is the first and only facility to date offering a user service program on cryoFIB/SEM. During the past year, eBIC has set up a workflow for microED of nanocrystals and thin crystal lamella. We hosted a microED workshop on practical data collection and data processing using DIALS in November 2019. A commissioning call for microED proposals will be announced once we are back to normal schedule. Additionally, eBIC director Dr Peijun Zhang gave 20 invited or keynote presentations at international meetings. Several eBIC scientists also presented at national and international meetings. To date, eBIC has produced 112 user publications, nearly doubled from the last report. The electron Physical Science Imaging Centre (ePSIC) ePSIC at Diamond is a national centre for aberration-corrected transmission electron microscopy. Since its opening in 2017, researchers from around the world have brought their samples to ePSIC to image their atomic structure with sub-ångström resolution. The two transmission electron microscopes which make up the centre, a JEOL ARM 200 and a JEOL GRAND ARM 300, were brought to Diamond through collaboration with Johnson Matthey and the University of Oxford respectively. The ARM 200 (E01) is a state-of-the-art probe-corrected analytical microscope capable of imaging, electron energy loss spectroscopy and X-ray spectroscopy at atomic resolution. It is aligned at accelerating voltages (incident electron beam energies) of 80 and 200 keV. The ARM 300 (E02) is a dedicated imaging instrument aligned across a wide range of accelerating voltages (30 - 300 keV). This enables the experimental conditions to be carefully tailored to the specific sample being studied. The ARM300 is both probe- and imaging-corrected and has numerous detectors including a fast direct electron detector, which can operate at up to 2000 fps. This detector is used for both fast movie acquisition when operating in broad- beam Transmission Electron Microscopy (TEM) mode and for the collection of large arrays of far-field diffraction patterns (4D-STEM) when operating in focused probe STEM mode. These combined capabilities have enabled ePSIC to become an international leader in cutting-edge material science electron microscopy and is a unique resource within the UK. ePSIC have recently added a Focused Ion Beam microscope (E03) and low energy ion slicer to its arsenal, enabling the preparation of incredibly thin samples – just a few-hundred atoms thick – for further analysis at ePSIC. This capabilitywill enable users, whomight otherwise have not been able to prepare the high quality samples required for atomic resolution imaging, to access ePSIC. With in situ sample holders, users at ePSIC can perform variable temperature measurements from 100 to 1,600 K to directly image the atomic structure of materials during thermally driven transitions. Staff at ePSIC have recently commissioned two new sample holders for in situ experiments. These new capabilities include the ability to image samples in the microscope while passing an electric current through them and the ability to transfer samples to the microscope without exposing them to air. 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 all around the world to work towards the visualisation of their membrane protein of interest at atomic resolution. Membrane proteins are important targets for medical, agricultural and fundamental research. Approximately half of all approved medicines target membrane proteins while some plant membrane proteins can be used to improve crop yield and resistance towards plant pathogens. Understanding the structure and function of these proteins helps us to develop newmedicines. As a Diamond facility located within the Research Complex at Harwell, and as an established member of the Harwell Cell and Structural Biology Partnership, MPL staff and visiting scientists are at the centre of structural biology on the Harwell campus. Traditionally we have grown membrane protein crystals, an extremely difficult step on the way towards solving a membrane proteins structure. Having a dedicated laboratory with cutting- edge equipment, close to the experimental stations where membrane protein structures can be solved, greatly enhances scientists’ ability to successfully crystallise membrane proteins and further our understanding of these important targets. Recently with the cryo-EM revolution, a new method has been opened up providing an alternative route towards solving the structure of these difficult to work with proteins. The close proximity of the MPL and eBIC is now enhancing our ability to understand the structures of membrane proteins by cryo-electron microscopy. We are currently in the process of recruiting a joint postdoctoral research associate across the MPL and eBIC to improve our capabilities further in the membrane protein and cryo-EM fields. 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 published work in Nature Communications 1 details research supported by MPL facilities that has led to the structure of ferric-enterobactin in complex with its transporter protein (PfeA) from Pseudomonas aeuginosa . Bacteria use small molecules called siderophores to scavenge and bind to iron. These complexes are imported into the periplasm through outer-membrane transporters such as PfeA. The crystal structure has been solved by the Naismith group of a mutant ferric entrerobactin receptor from P. aeruginosa and demonstrates that PfeA recognises enterobactin using extracellular loops distant from the pore. These results will provide starting points that could lead to the rational development of siderophore-antibiotic Trojan horse conjugates able to hijack more efficiently the enterobactin-dependent iron uptake system in P. aeruginosa . In Collaboration with the National Physics Laboratory and B23 beamline at Diamond Light Source research was published in the International Journal of Molecular Sciences 2 that discusses various biophysical methods for the characterisation of membrane proteins. This included characterisation of membrane protein-detergent complexes by in situ dynamic light scattering, multi-angle light scattering, Circular Dichroism of Membrane Proteins and Lipidic Cubic Phase Fluorescence Recovery After Photobleaching (LCP-FRAP). 1. Moynie. et al ., 2019. DOI:10.1038/s41467-019-11508-y 2. Kwan. et al ., 2019. DOI:10.3390/ijms20102605 MPL Team.