Diamond Concise Annual Review 2021/22

15 14 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 1 / 2 2 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 1 / 2 2 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 activity of the anticancer protein tubulin, the development of new antibiotics and determining the structure of a valuable protein used in neuroscience studies. Understanding the vital anticancer protein tubulin Tubulin plays an essential role in cell functions including cell division. Tubulin molecules form tube-like structures, called microtubule filaments, that give cells their shape and help transport proteins and other cellular components. Tubulin can bind to many proteins and small molecules, but the total number of binding sites was previously unknown. Swiss and Italian researchers investigated that question by using a unique combination of computer simulations and crystallographic fragment screening performed at the XChem facility and beamline I04-1. For the fragment screening, the team exposed hundreds of tubulin crystals to solutions containing fragments of molecules. Then, they used beamline I04- 1 to create X-ray diffraction patterns for each soaked crystal, showing which molecule fragments bound to the tubulin and where their binding took place. Their results uncovered 11 previously unknown binding sites on the protein and identified 56 fragments that bind to tubulin and could be used in future drug development. The team’s approach can also be used to investigate other proteins and could help to discover newbinding sites in other pharmaceutically important molecules. Mühlethaler T et al. DOI: 10.1002/anie.202100273 Designing effective new antibiotics Relatively fewantibioticswere introduced over recent decades, and overuse has led to the development of significant bacterial resistance. With some bacterial infections becoming untreatable, new antibiotic discovery is now a priority. An international teamof researchers in the UK and Slovenia designed a series of newmolecules with antibiotic potential. These new antibiotics, called Novel Bacterial Topoisomerase Inhibitors (NBTI's) kill bacteria and act against a well-validated target, DNA gyrase. By forming a complex with the enzyme and DNA, they stabilise single-strand DNA cleavage breaks, preventing the enzyme from functioning, which leads to cell death. To enable the design of further molecules, the team needed to know the molecular structure of the newmolecules bound to gyrase. They used beamline I04 to evaluate the relevant potency of several NBTIs against gyrase. In addition, they solved the crystal structure of gyrase bound to one of the new inhibitors, revealing the existence of bifurcated halogen bonds between the enzyme and the inhibitor molecule - an unprecedented observation in a biological system. This new biochemical and structural information informs the intelligent design of new molecules for use in clinical medicine. Kolarič A et al. DOI: 10.1038/s41467-020-20405-8 Developing new tools for neuroscience Archaerhodopsin-3 (AR3) is a light-sensitive protein expressed by the bacterium Halorubrum sodomense that is found in the Dead Sea bordering Israel and Jordan. Mutants of the protein are routinely used in neuroscience experiments to selectively silence individual nerve cells and detect changes in transmembrane voltage. However, these mutants are designed without knowledge of the protein’s structure. An international team of researchers were able to visualise the photoreceptor at unprecedented resolution using the I24 and B23 beamlines and reported the first ever structure of the ground state of AR3. The team was also able to crystallise the photoreceptor in a second conformation, a desensitised state that AR3 adopts in the prolonged absence of light. The superb resolution achieved for these AR3 structures is among the highest for a wild-type membrane protein deposited in the Protein Data Bank. The high-resolution crystal structures were essential for understanding the workings of the protein. These data provide structural biologists and protein engineers with the ‘blueprints’ to AR3, opening the way for the development of new tools and methodologies in the fields of neuroscience, cell biology and beyond. Bada Juarez JF et al . DOI: 10.1038/s41467-020-20596-0 VMXm beamline at Diamond