Diamond Annual Review 2020/21

30 31 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 Biological Cryo-Imaging Group MartinWalsh, Deputy Director of Life Sciences and Science Group Leader T heBiological Cryo-ImagingGroup brings together dedicated facilities for X-ray, light and electronmicroscopy at Diamond Light Source. The Electron Bio-Imaging Centre (eBIC) is the national centre for cryo-electron microscopy (cryo-EM) in the UK and provides a range of capabilities and supporting facilities for cryo-EM and Correlative Light and Electron Microscopy (CLEM). Beamline B24 hosts a full field cryo-transmission X-ray microscope dedicated to biological X-ray imaging and has also established a cryo super resolution fluorescence microscopy facility, which is a joint venture between Diamond and the University of Oxford. The group provides a unique platform for correlative light and X-ray microscopy, as well as cryo-EM. The Biological Cryo-Imaging (BCI) group has continued to grow and expand its capabilities during 2020 in spite of the COVID-19 pandemic. At eBIC, the team has expanded with a new Principal Electron microscopist, additional cryo-EM scientists and Post-Doctoral Researchers. At B24 we welcomed a new Beamline Scientist and a Post-Doctoral Researcher who is jointly funded by iNEXT-Discovery. In addition, the B24 team is hosting a Masters student from the University of Birmingham, funded through Diamond’s Year in Industry placement scheme. Remote access for both eBIC and B24 has been successfully established during 2020 and has quickly been taken up by our users. It has allowed us to sustain a user programme for supporting COVID-19 research during the first UK lockdown. Following on from these experiences, remote access has been extended to cover access to all instruments presently. At the time of writing, access to eBIC and B24 is still primarily through remote access, although as we come out of the current UK lockdown, users will be gradually welcomed back on-site. On 2 nd March 2020, Diamond published a rapid access call specifically for COVID-19 research proposals to ensure that we were doing everything possible to support researchers in their efforts to discover more about the virus and bring us closer to an effective vaccine or treatment. Although the UK went into its first lockdown in late March, the Diamond machine and eBIC microscopes were kept operating, albeit under reduced hours with core staff, to allow COVID-19 research to continue. This allowed us and our colleagues in Macromolecular Crystallography (MX) in particular to provide access and support to both internal and external groups working on COVID-19. At eBIC, a major focus of activity has been the spike (S) protein working in collaboration with groups at the Rosalind Franklin Institute and the University of Oxford. The first paper from this activity showed that lab-based production of novel potent single chain camelid antibodies (known as nanobodies) can successfully neutralise the SARS-CoV-2 virions 1 . The rapid characterisation of new variants, and the analysis of antibody binding to the Wuhan virus and its variants, has also been achieved at break-neck speed in a series of papers published in the journal Cell 2-5 , in which eBIC and the MX beamlines at Diamond have been instrumental in delivering the structural data. Combining cryo-electron tomography (cryo-ET) and X-ray tomography, in collaboration with the Central Laser Facility (CLF), Peijun Zhang’s group were able to track the SARS-CoV-2 assembly and egress pathway using correlative microscopy workflows that are being developed within the BCI group 6 . Zhang’s group are also collaborating with Sarah Gilbert, the lead researcher of the Oxford vaccine team, and have applied cryo-ET to characterise the structure of the spike (S) proteins generated by the Oxford/Astra Zeneca vaccine. They have confirmed the use of the technology for SARS-CoV-2 vaccines by showing that the structures of the expressed S proteins on the surface of cells mimicked that of cells infected with SARS-CoV-2 7 . At B24, in addition to collaborating with eBIC on visualising the life cycle of the SARS-CoV-2 virus in a cellular context, the beamline has also been used through Diamond’s industry access to help fast track new diagnostic tests for COVID-19. On the instrument front at eBIC, a new partnership with Diamond and the Research Complex at Harwell (RCaH) has resulted in eBIC hosting a new200 keV Thermofisher Scientific Glacios cryo transmission electron microscope. This has been made possible through funding by UKRI-Medical Research Council (MRC) to the RCaH. eBIC has integrated the microscope into the facility and is working with RCaH to provide a user and training programme to Harwell campus-based groups. On the correlative side at eBIC, we have finalised commissioning of our recently purchased Leica cryo Correlative Light and Electron Microscope (cryo- CLEM) and published a user commissioning call in spring of 2021. Finally, eBIC have worked extensively with the manufacturer Thermofisher to commission and benchmark their prototype energy filter - Selectris. Performance and stability tests showed the Krios hall to be an exceptionally stable environment. The Selectris filter has been used preferentially by many user groups for COVID-19 related projects in cryo-EM and cryo-ET, where the unparalleled stability and performance have enabled many novel observations. Full access is now available to all eBIC Rapid Access and BAG users. At B24, we have streamlined the cryo Correlative Light and X-ray Tomography (cryo-CLXT) workflow with new protocols for sample and data correlation. In addition, in the last year at B24 a huge effort has gone into detailing all our protocols, with major updates to our webpages and publication of the protocols in a series of articles 8-10 . Training continues to be a major focus for the group and in spite of the lockdown, we have been able to successfully carry out and plan remote training across a wide audience. Workshops at eBIC include an Instruct-ERIC sponsored workshop on fully remote data collection, which took place in May 2021, while at B24 the team were involved in, and contributed to, an iNEXT-Discovery two-day Zoom workshop focused on the basics, pros/cons and opportunities in using the techniques of cryo-ET and cryo soft X-ray tomography to answer biological questions. The B24 user base were also provided with intensive training to take full advantage of remote access that is now implemented at the beamline. It has been a very different year and as always, what we have achieved would not have been possible without the dedication of our scientific staff, technicians, software and engineering teams who support biological cryo- imaging, and the administrative and infrastructure support groups across Diamond, our colleagues at RCaH, The Franklin and our users! References: 1. Huo J. et al. Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2. Nat. Struct. Mol. Biol. 27 , 846–854 (2020). DOI: 10.1038/s41594-020-0469-6 2. Dejnirattisai W. et al. The Antigenic Anatomy of SARS-CoV-2 Receptor Binding Domain. SSRN Electron. J. (2021). DOI: 10.2139/ssrn.3754542 3. Dejnirattisai W. et al. Antibody evasion by the P.1 strain of SARS-CoV-2. Cell (2021). DOI: 10.1016/j.cell.2021.03.055 4. Supasa P. et al. Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera. Cell 184 , 2201-2211.e7 (2021). DOI: 10.1016/j.cell.2021.02.033 5. Zhou D. et al. Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera. Cell 184 , 2348-2361.e6 (2021). DOI: 10.1016/j.cell.2021.02.037 6. Mendonça L. et al. SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging. bioRxiv 2020.11.05.370239 (2020). DOI: 10.1101/2020.11.05.370239 7. Watanabe Y. et al. Native-like SARS-CoV-2 Spike Glycoprotein Expressed by ChAdOx1 nCoV-19/AZD1222 Vaccine. ACS Cent. Sci. 7 , 594–602 (2021). DOI: 10.1021/acscentsci.1c00080 8. Iudin A. et al. Data-deposition protocols for correlative soft X-ray tomography and super-resolution structured illumination microscopy applications. STAR Protoc. 2 , 100253 (2021). DOI: 10.1016/j. xpro.2020.100253 9. Kounatidis I. et al. 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway. Cell 182 , 515-530.e17 (2020). DOI: 10.1016/j. cell.2020.05.051 10.Phillips M. A. et al. CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging. Optica 7 , 802 (2020). DOI: 10.1364/optica.393203 Serial cryoFIB/SEM volume imaging of entire SARS-CoV-2 infected cell. Shown are various views of a surface rendering of the segmented volume of SARS-CoV-2 infected cell. Segmented organelles and virus particles are labelled with colours indicated. Figure adapted fromMendonça et al. (2020). SARS-CoV-2 cytoplasmic viral assembly (Figure reproduced fromMendonça et al. (2020). (A-B) CryoFIB/SEM images of two sequential slices separated by 80 nm. Black arrows point to virus particles in single membrane vesicle (SMV). Pink arrow points to small dense vesicles lining the outside of virus-containing SMV. (C) Tomographic slice of cryoFIB lamella depicting SARS-CoV-2 assembly, with DMV portals (yellow arrow), assembling viruses (blue arrow), assembled virus (black arrow), viral spikes on SMV membranes (red arrows), dense vesicles around the assembly site (pink arrow, as in B) and a nucleopore (black arrowhead). (D) Density segmentation of C, displaying three virus particles (black arrows) and two assembly sites (blue arrows). (E) An enlarged view (at a different angle) of boxed area in C, showing assembled virus (black arrow), assembling viruses (blue arrows), spikes (red arrows) and spike-containing vesicles (pink arrows). (F) Large intracellular virus-containing vesicle (LVCV) full of readily assembled viruses. (G) Subtomogram average of viral spikes of intracellular viruses from cell lamellae at 11 Å resolution, fitted with an atomic model of spike trimer (PDB 6ZB5) (Toelzer et al., 2020). Scale bar is 300 nm in A and B; and 100 nm in C, E and F. Newly recruited cryo-EM scientist, David Owen, working on the RCaH Glacios transmission cryo-electron microscope, which has been recently installed at eBIC and will be run in partnership with RCaH.