Diamond Concise Annual Review 2020/21

16 17 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 T his group brings together dedicated facilities for X-ray, light and electron microscopy 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-EMand Correlative Light and ElectronMicroscopy (CLEM). BeamlineB24hosts 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. Studies undertaken this year include new understanding of the structure and function of cytotoxic proteins which protect the body from cancers and infections, pioneering design on newantibiotic therapies, and structure-based design of drugs with reduced side effect profiles. New insights on our immune system The white blood cells called cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells are components of the immune system that help cure viral infections and prevent the progression of cancers. CTL and NK cells kill infected cells by secreting cytotoxic proteins (so-called ‘protein bombs’) onto their surface. An international team of researchers interested in new biological therapies for cancer set out to examine how these proteins avoid dilution and enter target cells. The team used Diamond Light Source's beamline B24 with low-energy (soft) X-rays to generate 3D maps of organic material in cells and small particles released from cells. Soft X-rays allow imaging of entire T cells, although the resolution is lower than when using high-energy X-rays. The results allowed the team to confirm that the ‘protein bombs’ are packaged in supramolecular attack particles (SMAPs) which contain more than 280 kinds of protein. They also demonstrated detailed structures and identified potential storage sites for the ‘protein bombs’ in theT cells.This synchrotron technology shows great promise in the study of cellular structures and how T cells protect the body and can be used to further improve cancer therapies. Balint S. et al. DOI: 10.1126/science.aay9207 Designing new therapies Bacteria use a wide range of trans-membrane secretion systems to transfer proteins into target cells. These ‘bacterial effector proteins’ interfere with host cell functions and could have important applications in designing new antibiotics. An international team of researchers worked to understand the mechanism by which Legionellapneumophila (which causesLegionnaires’disease) uses a secretion system to infect human cells. They were able to determine the atomic resolution structure of the system using single-particle cryo-EM with data collected at eBIC at Diamond Light Source. This showed a five-protein core complex which combines with others to form a large nanomachine, containing a channel in the membrane, that protein effector molecules can pass through. Analysis of multiple cryo-EMmaps, further modelling and mutagenesis provided working hypotheses for the mechanism of binding and delivery of two classes of Legionella effectors. Gaining a better understanding of how secretion systems work and resolving the high-resolution structure of the Legionella secretion system provides essential information to help design antibiotics that block the system and could also provide the basis for engineering secretion systems capable of injecting therapeutic drugs into human cells. Meir A. et al. DOI: 10.1038/s41467-020-16681-z Designing drugs with reduced side effects The human body relies on an extensive network of signalling molecules (agonists) such as hormones to coordinate bodily functions. Receptors on the surface of cells bind these hormones and activate a signalling cascade to alter the cell’s biochemistry. G protein-coupled receptors (GPCRs) are the largest and most diverse family of these receptors and therefore many therapeutic drugs have been designed to target them. GPCRs have two signalling pathways (the G protein-coupled pathway and the arrestin-mediated pathway) and, in many cases, one is the therapeutic pathway while the other may produce side effects. If a drug could be developed that could signal only down the therapeutic pathway (a ‘biased agonist’), then side effects could be significantly reduced. To investigate this, an international research team used cryo-EM at eBIC at Diamond Light Source to determine the structure of a GPCR coupled to arrestin and compare its structure to the G protein-coupled state, with the same agonist bound to both receptors.They identified two regions of the GPCR that could be used in the development of ‘biased’ agonists and the study provides valuable data for structure-based drug design. Lee Y. et al. DOI: 10.1038/s41586-020-2419-1 Biological Cryo-Imaging Group Image from a tomogram of a white blood cell interacting with an electron microscopy grid and highlighting a multicore granule (arrow).