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Every year, Diamond produces an Annual Review, covering the scientific, technical, computing and business updates from the facility. The feature that follows has been prepared for our latest review, and looks at work conducted between April 2023 to April 2024.
The 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 from crystalline samples of 1 μm and upwards currently. A wide 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 a wide 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 250,000th 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. The capability of crystallographic fragment screening available at Diamond was illustrated exquisitely during the COVID-19 pandemic with several proteins targeted for investigation. This was exemplified by the COVID Moonshot programme and it’s targeting of the main protease for antiviral inhibitors. The data was made available immediately to the science community, and the methods and results of an open science, crowdsourced and structure based drug discovery campaign have recently been published1
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 elements2, 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 chlorine3. To optimise and improve data quality the I23 team has established an analytical absorption correction based on X-ray tomography4 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.
Beamline I24 is a fully tuneable, high flux microfocus beamline that enables both ‘standard’ cryo MX 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 and multiplex 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 serial crystallographic data. This offers the ability to both assess microcrystals that appear in vapour diffusion plates and to assess the quality of batch crystallisation experiments by transferring small aliquots to a crystallisation plate for grid scanning. This work is described in Thompson et al. (2024) Acta Cryst. D. 80, 279-2885.
The micro/nanofocus beamline VMXm has had a successful year taking users to assist with our optimisation of the beamline. Some novel structures including that from a 70-year-old crystal sample (Keown et al6) have resulted, and interest from both academic groups and industry is growing for both macromolecular and small molecule research.
The high-energy electron Xtallography Instrument (HeXI) funded by Wellcome will further enhance the MX group’s capabilities to perform micro to nano crystal diffraction analyses of protein and small molecule crystals. The HeXI team has recently completed the Technical Design Review for the phase two instrument and this has enabled the project to successfully tender for the MeV electron source. The full instrument layout will also enable the imminent issuing of the tender for the experimental hutch. The commencement of the procurement of these two high-value, long lead-time items are highly significant project milestones towards the anticipated successful delivery of the instrument in 2026.
Since April 2023, the Advanced Photon Source, USA has been undergoing a machine upgrade to its fourth generation source APS-U and Diamond MX collaboratively have been supporting the APS MX academic user community. APS have run several user calls for MX access to Diamond and Diamond have been providing remote access and UDC to MX beamlines I03, I04, I04-1 and I24. This will continue through until September 2024.
The MX group continued to offer a wide range of outreach and training opportunities over the last year. These included our regular two-day BAG trainings to share updates and best practices at the beamlines, laboratories and our software, sample preparation for serial synchrotron crystallography and XChem training visits. We celebrated the 10th year of running the joint CCP4-Diamond crystallography workshop in December with over 20 early career researchers coming to Diamond for nine days to work closely with experts in the field to analyse their own data and collect data at Diamond’s MX beamlines. This year we have initiated outreach workshops to showcase the science capabilities of the beamlines and facilities with our first event held at the Francis Crick Institute and further workshops around the UK are in the pipeline.
In 2023, MX group users published nearly 300 journal papers and deposited nearly 1,200 structures in the publicly accessible protein data bank and the MX group underwent a successful international review for Diamond’s Science Advisory Committee. The scientific output for our users and the capabilities available to them is thanks to the expertise, efforts and dedication of the staff of the MX group and the many support groups that help deliver a user programme optimised around user needs alongside continual development to bring improved and new capabilities regularly to the user community.
1. Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors
2. Experimental phasing opportunities for macromolecular crystallography at very long wavelengths
3. High-Confidence Placement of Fragments into Electron Density Using Anomalous Diffraction—A Case Study 4. Using Hits Targeting SARS-CoV-2 Non-Structural Protein 1
5. Ray-tracing analytical absorption correction for X-ray crystallography based on tomographic reconstructions
6. Efficient in situ screening of and data collection from microcrystals in crystallization plates
7. Atomic structure of a nudivirus occlusion body protein determined from a 70-year-old crystal sample
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