Diamond Light Source - Annual Review 2022/23 - Concise Edition

19 18 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 2 / 2 3 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 2 / 2 3 Macromolecular Crystallography Group M acromolecular crystallography (MX) exploits the hard energy, high flux X-rays created at Diamond Light Source to enable our user community to investigate the structure and function of biological macromolecules at atomistic resolution and up to millisecond timescales. This provides deep insight into the details of biological activity key to our understanding of the processes of life. Diamond provides access to a suite of seven MX beamlines (I03, I04, I04-1, I23, I24, VMXi and VMXm) to a large international academic and industrial user community. The beamlines cover a very broad range of capabilities from high throughput, micro- and nano-focus beams, extremely long wavelengths, room temperature in situ collection from crystallisation plates, (time resolved) serial synchrotron crystallography (SSX), a fragment-based screening platform (XChem) and the Membrane Protein Laboratory. Important research studies conducted this year included new insights into the role of inositol in gut bacteria, the development of new therapeutic nucleic acids and a new software developed at Diamond to solve structure base onmulti crystals analysis. Understanding the biochemistry of gut bacteria Lipids are vital components of all cells, forming the main component of cell membranes. Many different lipids are found in membranes, with a wide range of functions. Inositol is a carbocyclic sugar that is a key player in eukaryotic cells and forms the polar head group of inositol lipids. Inositol lipids are not major components of eukaryotic cell membranes, but they play important roles in cell division and signalling between cells. While inositol lipids are widely distributed across eukaryotes, little is known of their role and structure in bacteria. An international team of researchers investigated how the dominant gut microbe Bacteroides thetaiotaomicron makes inositol lipids. B. thetaiotaomicron is an importantmember of the human gutmicrobiome and can use awide range of dietary carbohydrates as carbon sources. After identifying the genes responsible for B. thetaiotaomicron inositol lipid synthesis, the researchers deleted each of these from the genome to see their effect on the production of the inositol lipids. B. thetaiotaomicron bacteria with genomic deletions in the inositol lipid synthesis genes are less able to survive in their host. Changes in the composition of the gut microbiome can affect host health. By developing an understanding of the biochemistry of bacteria in the gut, we can gain knowledge of the roles they play in various diseases, such as inflammatory bowel diseases. Heaver, SL, et al. DOI: 10.1038/s41564-022-01152-6 Structural studies of the enhanced binding affinity of therapeutic nucleic acids to proteins Introducing phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids significantly increases their stability and potency. The phosphorothioate backbone is the most widely used modification in therapeutic nucleic acids, including antisense oligonucleotides (ASOs). This modification involves a replacement of one of the two oxygen atoms in the repeating phosphate groups of the DNA with sulphur. PS-modified nucleic acids show improved properties, such as metabolic stability from nuclease-mediated degradation. The molecular mechanisms of interactions between PS nucleic acids and proteins have not been fully established. To better understand how PS ASOs interact with cellular proteins, researchers used the I23 beamline to solve two crystal structures of PS ASO bound to annexin A2 (AnxA2), a calcium-binding protein previously implicated in the release of PS ASOs from endo-lysosomal compartments. Their results unambiguously confirmed, for the first time, that van derWaals contacts between the sulphur atom and hydrophobic parts of arginine and lysine side chains are the driving force for enhanced interaction of PS ASOwith proteins. Overall, their results provide valuable insights into the general mechanism of the enhanced binding of PS ASOs to cellular proteins and indicate that the interaction between PS linkages and lysine and arginine residues is a general phenomenon that is observed not only for nucleic acid-binding proteins but may also account for the association of ASO with proteins that are not known to bind DNA. This work provides information that will be instrumental in the rational design of improved nucleic acid-based drugs. Hyjek-Składanowska M. et al . DOI: 10.1093/nar/ gkac774 xia2.multiplex - a newpipeline for multi-crystal data analysis A team of scientists at Diamond has developed a new program, xia2. multiplex, to facilitate and help to optimise the scaling and merging of multiple data sets. At the very beginning of macromolecular crystallography, structures were obtained using room temperature data collection, and it was common to merge together data from multiple crystals to obtain a complete data set. With the increasing availability of synchrotron sources with ever more intense beams in the 1990s, radiation damage was becoming a limiting problem, leading to the popularisation of “cryogenic” data collection. Such flash-cooling of crystals to around 100 K significantly extends the crystal lifetime in the X-ray beam. In recent years, there has been an increasing awareness that cryocooling can “hide” biologically significant structural features. So there has been interest in collectingdataundermorephysiologically relevant roomtemperature conditions. In addition, many scientifically important targets, such as membrane proteins and viruses, frequently yield small, weakly diffracting microcrystals. Improvements in beamline, detector technology and experimental techniques have made collecting data from such crystals more tractable. However, data processing remained challenging compared to the user experience for more routine experiments andmultiple crystals are often required even at 100 K. The research team has demonstrated that xia2.multiplex can be used to combine multi-crystal datasets. Its implementation within the wider MX data analysis pipelines makes it readily available to MX users at Diamond, providing them with timely feedback on multi-crystal experiments. xia2.multiplex can be applied to a wide variety of multi-crystal datasets, from multi-crystal phasing experiments on I23 to room temperature in situ data collections on I24 andVMXi. Gildea, RJ. et al DOI 10.1107/S2059798322004399