16 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 BeamlineVMXi xia2.multiplex - a newpipeline formulti-crystal data analysis Pathogens – Infectious Diseases – Health &Wellbeing – Information & Communication Technologies – Structural Biology – Data processing – Life Sciences & Biotech 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 on weak (in comparison to synchrotrons) laboratory X-ray sources, 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 (i.e. themaximumX-ray dose that a crystal can tolerate) 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, thereby increasing the quantity of data that can be collected per crystal. As a result, most macromolecular structures obtained over the last 20-30 years were solved using data from a single crystal under cryogenic conditions. Simultaneously, the development of highly-automated data analysis pipelines such as xia2 and fast_dp has made macromolecular crystallography more accessible to structural biologists, enabling more efficient use of beamtime for routine experiments. However, while undoubtedly successful, cryocooling has a number of limitations. In recent years, there has been an increasing awareness that cryocooling can “hide” biologically significant structural features. So there has been interest in collecting data under more physiologically-relevant room temperature conditions (with the trade off that significantly less data can be obtained froma single crystal). Certain classes of macromolecular crystals, such as viruses, can suffer damage on cryocooling, necessitating room temperature data collection for improved data quality. In addition, many scientifically important targets, such as membrane proteins and viruses, frequently yield small, weakly diffracting microcrystals. Improvements in beamline (more intense, microfocus beams) and detector (speed and sensitivity) 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 and multiple crystals are often required even at 100 K. Combining incomplete datasets from multiple crystals poses several challenges. Firstly, determining an overall symmetry can be challenging when combining sparse datasets of varying quality. This problem is addressed by a new approach to symmetry determination implemented in the Diamond developed software dials.cosym and used by xia2.multiplex. Other challenges inmulti-crystal data collections include the identification and rejection of “non-isomorphous” or poor-quality datasets from particular crystals and the assessment of the levels of possible radiation damage, particularly for room temperature datasets. Whilst this software development applies to all MX beamlines, it is particularly relevant to I24, VMXi and VMXm, where room temperature experiments or data collections onmicro-crystals or room temperature crystals within their crystallisation droplet typically necessitate combining data from multiple crystals. 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 and VMXi. It is expected to play a critical role in future research investigating room temperature fragment screening on VMXi as part of the EU-funded “Fragment-Screen”project. https://instruct-eric.org/news/fragment-screen-a-new-european- project-coordinated-by-instruct-eric/ Related publication: Gildea, RJ. et al. xia2. multiplex: a multi-crystal data-analysis pipeline. Acta Crystallographica Section D: Structural Biology 78 , 6: 752-769 (2022). DOI 10.1107/S2059798322004399 Funding acknowledgement: STFC via CCP4 Biostruct-X project No. 283570 of the EU FP7 theWellcome Trust (grant No. 202933/Z/16/Z and 218270/Z/19/Z) US National Institutes of Health grants GM095887 and GM117126 Corresponding authors: Dr Richard Gildea, Diamond Light Source,
[email protected] Room temperature ligand binding results using data from 8 crystals of SARS CoV2 macrodomain 1 (MAC1) collected at VMXi and merged together with xia2.multiplex. a) Evidence at the ligand binding site for the presence of the ligand (green mesh); b) Ligand included within the structure to show interactions with the protein; C) overall structure of the protein with the ligand binding site shown. 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 2 / 2 3 Macromolecular Crystallography Group Beamline I24 Freeze-frame views of proteins inmotion: howdo acid and alkaline conditions alter the structure of a fluorescent protein? Physical Chemistry – Biochemistry – Chemistry – Structural Biology – Life Sciences & Biotech Proteins are the building blocks of life – our entire body is made from them, including structural proteins, enzymes, hormones and antibodies. X-ray crystallography is a well-established technique for determining the 3D structure of a protein crystal, which helps us to understandhowproteins function. Serial Synchrotron Crystallography (SSX) is an emerging method that can capture a series of snapshots of proteins during motion and piece them together, like a stop-motion movie, to understand the critical steps and components in the protein’s activity. This allows movies of protein motion to be captured on timescales spanning femtoseconds to seconds with resolution high enough to observe the movements of single atoms in the protein structure. A wide array of reversibly photoswitchable fluorescent proteins (rsFPs) have been developed and are used across microscopy in biological sciences to track cell behaviour, develop bioelectronics and image living things. A key aspect of reversibly switchable fluorescent proteins is their ability to switch ‘on’ and ‘off’. However, in low pH (acidic) conditions, this switching technique is poorly understood. A team of researchers from Imperial College London conducted experiments to increase our understanding of the switching reactions in these proteins at low pH and compare it to neutral pH to see if there were differences in switching mechanisms. Diamond’s I24 beamline provided a bespoke set-up to capture freeze frames of the protein in motion, which the researchers combined with infrared spectroscopy measurements made in their own lab. Obtaining the structure at specific pH levels and illumination conditions allowed them to directly correlate particular structures to the infrared signals they measured. Using these techniques, the team discovered that at acidic pH, the photo-switching behaviour of their protein followed a different cycle to that at an alkaline pH. They also made the first observation of switching to a new chromophore charge state. Experiments like this one demonstrate that the combination of synchrotron microfocus beams and serial crystallography is a powerful one. Working together, Diamond and Imperial were able to develop existing techniques into a new set of tools to investigate a particular biological problem. Their results will directly inform the design of new reversibly switchable fluorescent proteins, especially with red-shifted emission spectra. It could also form the basis for new ultrafast measurements to study pathways of the photoexcitation in these samples. Related publication: Baxter, JM. et al. Observation of cation chromophore photoisomerization of a fluorescent protein using millisecond synchrotron serial crystallography and infrared vibrational and visible spectroscopy. The Journal of Physical Chemistry B 126, 45 9288-9296 (2022). DOI:10.1021/acs.jpcb.2c06780. Funding acknowledgement: EPSRC studentship BBSRC (grant BB/P00752X/1) Imperial College Centre for Structural Biology Corresponding authors: Jasper van Thor, Imperial College London,
[email protected] James M Baxter, Columbia University,
[email protected] At pH below the chomophore pKa, the photocycle of an engineered fluorescent protein includes the first observation of photoisomerization of the cation state.