Diamond Annual Review 2023/24

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 3 / 2 4 Macromolecular Crystallography Group 1 https://www.science.org/doi/10.1126/science.abo7201 2 https://www.nature.com/articles/s42004-023-01014-0 3 https://www.mdpi.com/1422-0067/24/13/11197 4 https://journals.iucr.org/j/issues/2024/03/00/yr5123/index.html Dave Hall, Science Group Leader T he macromolecular crystallography (MX) group at Diamond supports a range of instruments and facilities that support UK and worldwide structural biologists from both academia and industry. The science MX supports is broad, encompassing disease, ageing, food security, biotechnology, microbial drug resistance and vaccine and drug development. Seven X-ray beamlines (I03, I04, I04-1, I23, I24, VMXi and VMXm), the UK XFEL-Hub, XChem and membrane protein laboratories support one of the broadest ranges of experimental capabilities anywhere for investigating structural and temporal biological information from macromolecules. In development is the high energy electron diffraction instrument HeXI to further complement the suite of facilities. High quality, high-resolution three-dimensional structures can be obtained fromcrystalline samples of 1 µmand upwards currently. Awide range of available X-ray energies (2.1 – 28 keV) can be used to elucidate the exact atomic structures of molecules of interest and pinpoint awide range of ions and atoms. Data collection at room temperature in a variety of media and delivery systems can illustrate crystal quality for feedback into further experiments, provide complementary near physiological temperature structures and near zero dose structures. Reaction mechanisms down to millisecond time regimes can be investigated at our beamlines and down to femtoseconds via our community support of access to XFELs via the UK XFEL-Hub. The uptake of unattended data collection (UDC) implemented on the three high throughput beamlines I03, I04 and I04-1 continued to increase across the last year. Of note, in December 2023 the 250,000 th sample was investigated through this method of fully automated data collection since introducing it in June 2020. With the Diamond-II project on the horizon and expected roll-out of a new software stack, UDC on I03 is being used to prototype and develop this next generation of software on an operational beamline. This development enhances capabilities for current users at the same time as preparing for the ultra high throughput beamline K04 for drug discovery and general user programme that will open with the new source. On I04 the combination of microfocus and variable focus beam in combination with multi-axis goniometry and the available software functionality provides a toolkit to address a vast range of macromolecular data collections including very challenging samples and addressing all experimental aims in cryocrystallography. in the last year, the X-ray source has been optimised further, improving the flux profile and extending the beamline energy range (6 – 20 keV) coupled with an increase in flux enabling routine use of the detector at its fastest acquisition rate of 500 Hz. The dose tool has been further developed, users have been trained in its use and as a result it is now used by 90% of the interactive users providing optimised parameters for data collection. The team also encourages and supports in-person visits where in-depth training is provided. Beamline I04-1 continues to support experiments driven by the users of the fragment based drug discovery (FBDD) laboratory XChem (see Integrated Facilities section). The capability of crystallographic fragment screening available at Diamond was illustrated exquisitely during the COVID-19 pandemic with several proteins targeted for investigation, exemplified by the COVID Moonshot programme and it’s targeting of the main protease for antiviral inhibitors which whilst data was made available immediately to the science community, the methods and results of an open science, crowdsourced and structure based drug discovery campaign have recently been published 1 . At Diamond-II, the XChem programme will move to a wholly new beamline K04 which aims to automate the process of FBDD at Diamond. The team have worked through the conceptual design and technical designs towards this project are now well advanced. The long wavelength beamline I23 has continued to establish itself as the worldwide unique instrument for MX in a wavelength range not available elsewhere. With its in vacuum sample environment and a detector covering a large angular range it serves for a wide range of experiments. Whilst originally designed for phasing experiments from intrinsically bound elements 2 it has now transitioned into exploiting its unique design such that users can locate and differentiate biologically relevant elements such as calcium, potassium, chlorine, sulfur, phosphorous, magnesium and sodium in macromolecules. This enables not only a clear understanding of the position and roles of these elements in biological molecules but also can assist model building at low resolution and placement of ligands such as fragments in drug discovery campaigns which often contain anomalous scatterers such as sulfur or chlorine 3 . To optimise and improve data quality the I23 team has established an analytical absorption correction based on X-ray tomography 4 and laser ablation to shape samples and remove non-diffracting materials. In addition to understanding the role of ions in biology, the beamline is also able to collect very high resolution data. All taken together the continued improvements on I23 and in particular with the laser shaping set-up have allowed to grow its user community from both academia and industry who are interested in applying its unique capabilities. I24 is a fully tuneable, high flux microfocus beamline that enables both ‘standard’cryoMX and serial synchrotron crystallography (SSX).The community of users exploiting SSX at I24 continues to grow. Users benefit from a range of flexible sample delivery options, coupled with either laser or substrate reaction triggering. Serial data and multi-crystal data are processed automatically by xia2.ssx andmultiplex respectively. Future developments to I24 will be realised through rolling improvements as part of the I24 KMX flagship project. These include an improved large area detector, multilayer monochromator and hardware for improved beam focussing and stability. These upgrades will have both an immediate impact on the ongoing user programme and in future will enable I24 to fully exploit the gains offered by the Diamond II upgrade. The room temperature beamline VMXi now offers the ability to measure grid scans from crystallisation droplets and to treat the resulting data as