X-ray characterisation methods have undoubtedly enabled cutting-edge advances in all aspects of m... more X-ray characterisation methods have undoubtedly enabled cutting-edge advances in all aspects of materials research. Despite the enormous breadth of information that can be extracted from these techniques, the challenge of radiation-induced sample change and damage remains prevalent. This is largely due to the emergence of modern, high-intensity X-ray source technologies and growing potential to carry out more complex, longer duration in-situ or in-operando studies. The tunability of synchrotron beamlines enables the routine application of photon energy-dependent experiments. This work explores the structural stability of [Rh(COD)Cl] 2 , a widely used catalyst and precursor in the chemical industry, across a range of beamline parameters that target X-ray energies of 8 keV, 15 keV, 18 keV and 25 keV, on a powder X-ray diffraction synchrotron beamline at room temperature. Structural changes are discussed with respect to absorbed X-ray dose at each experimental setting associated with the respective photon energy. In addition, the X-ray radiation hardness of the catalyst is discussed, by utilising the diffraction data at the different energies to determine a dose limit, which is often considered in protein crystallography and typically overlooked in small molecule crystallography. This work not only gives fundamental insight into how damage manifests in this organometallic catalyst, but will encourage careful consideration of experimental X-ray parameters before conducting diffraction on similar radiation-sensitive organometallic materials.
Knowledge of protein structures is of central importance in modern drug discovery and molecular b... more Knowledge of protein structures is of central importance in modern drug discovery and molecular biology, but to be useful the structures, including those obtained in the crystalline state, must be biologically relevant. Small variations in crystallisation conditions can lead to alternative crystal forms, conformations and oligomerization states, causing changes which can lead to altered fold and active site architectures. In the determination of protein structures by Xray crystallography, crystallisation is an essential prerequisite and remains a major bottleneck in the drug discovery. Although many methods have been tried in an attempt to improve the production of protein crystals, it is still largely a 'trial and error' process. To our knowledge, crystallisation by cross-seeding using homologous proteins has previously only been successful for proteins with greater than 61-74% sequence identity. In the study presented here, we explore the effect of low sequence similarity, but fold homology, ??? on cross seeding using metallo-βlactamases with sequence identities as low as 19% and sequence similarity of only 36%, byt wth homologous core folds. Despite the low sequence identities, the results show that Micro-Cross-Seeding Matrix Screening (MCMS) can increase the number of hits obtained, and shorten crystallization time. It can also help in the identification of new crystallization conditions and different crystal forms.
Traditionally small-molecule crystallographers have not usually observed or recognized significan... more Traditionally small-molecule crystallographers have not usually observed or recognized significant radiation damage to their samples during diffraction experiments. However, the increased flux densities provided by third-generation synchrotrons have resulted in increasing numbers of observations of this phenomenon. The diversity of types of small-molecule systems means it is not yet possible to propose a general mechanism for their radiation-induced sample decay, however characterization of the effects will permit attempts to understand and mitigate it. Here, systematic experiments are reported on the effects that sample temperature and beam attenuation have on radiation damage progression, allowing qualitative and quantitative assessment of their impact on crystals of a small-molecule test sample. To allow inter-comparison of different measurements, radiation-damage metrics (diffraction-intensity decline, resolution fall-off, scaling B-factor increase) are plotted against the absorbed dose. For ease-of-dose calculations, the software developed for protein crystallography, RADDOSE-3D, has been modified for use in small-molecule crystallography. It is intended that these initial experiments will assist in establishing protocols for small-molecule crystallographers to optimize the diffraction signal from their samples prior to the onset of the deleterious effects of radiation damage.
Much progress has been made over the last 15 years in characterising radiation damage (RD) to mac... more Much progress has been made over the last 15 years in characterising radiation damage (RD) to macromolecular crystals during 100 K X-ray diffraction experiments [1], and to a lesser extent, for those irradiated at room temperature (RT). Despite a now extensive body of literature on various aspects of RD in MX and SAXS (e.g.
X-ray characterisation techniques are invaluable for probing material characteristics and propert... more X-ray characterisation techniques are invaluable for probing material characteristics and properties, and have been instrumental in discoveries across materials research. However, there is a current lack of understanding of how X-ray induced effects manifest in small molecular crystals. This is of particular concern as new X-ray sources with ever increasing brilliance are developed. In this paper, systematic studies of X-ray-matter interactions are reported on two industrially important catalysts, [Ir(COD)Cl] 2 and [Rh(COD)Cl] 2 , exposed to radiation in X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) experiments. From these complimentary techniques, changes to structure, chemical environments, and electronic structure are observed as a function of X-ray exposure, allowing comparisons of stability to be made between the two catalysts. Radiation dose is estimated using recent developments to the RADDOSE-3D software for small molecules and applied to powder XRD and XPS experiments. Further insights into the electronic structure of the catalysts and changes occurring as a result of the irradiation are drawn from density functional theory (DFT). The techniques combined here offer much needed insight into the X-ray induced effects in transition metal catalysts and consequently, their intrinsic stabilities. There is enormous potential to extend the application of these methods to other small molecular systems of scientific or industrial relevance.
Understanding mechanisms of antibody synergy is important for vaccine design and antibody cocktai... more Understanding mechanisms of antibody synergy is important for vaccine design and antibody cocktail development. Examples of synergy between antibodies are well-documented, but the mechanisms underlying these relationships often remain poorly understood. The leading blood-stage malaria vaccine candidate, CyRPA, is essential for invasion of Plasmodium falciparum into human erythrocytes. Here we present a panel of anti-CyRPA monoclonal antibodies that strongly inhibit parasite growth in in vitro assays. Structural studies show that growth-inhibitory antibodies bind epitopes on a single face of CyRPA. We also show that pairs of non-competing inhibitory antibodies have strongly synergistic growth-inhibitory activity. These antibodies bind to neighbouring epitopes on CyRPA and form lateral, heterotypic interactions which slow antibody dissociation. We predict that such heterotypic interactions will be a feature of many immune responses. Immunogens which elicit such synergistic antibody mixtures could increase the potency of vaccine-elicited responses to provide robust and long-lived immunity against challenging disease targets.
Radiation damage inflicted on macromolecular crystals during X-ray diffraction experiments remain... more Radiation damage inflicted on macromolecular crystals during X-ray diffraction experiments remains a limiting factor for structure solution, even when samples are cooled to cryotemperatures (100 K). Efforts to establish mitigation strategies are ongoing and various approaches, summarised below, have been investigated over the last 15 years, resulting in a deeper understanding of the physical and chemical factors affecting damage rates. The recent advent of X-ray free electron lasers permits 'diffraction before destruction' by providing highly brilliant and short (a few tens of fs) X-ray pulses. New 4 th generation synchrotron sources now coming on line with higher X-ray flux densities than those available from 3 rd generation synchrotrons, will bring the issue of radiation damage once more to the fore for structural biologists. Keywords X-ray-matter interactions, global and specific radiation damage, radicals and their scavengers, absorbed dose, radiation damage mitigation, cryocrystallography Running head Radiation damage 'diffraction before destruction' with no significant radiation damage. The new methodology associated with these experiments is covered elsewhere in this book in the chapter by Chapman. The physics involved in radiation damage is well understood and characterized, although the same cannot be said about the chemical aspects. When an X-ray beam of the energy range usually used for MX (6-15 keV) is incident on a sample, it interacts by three main mechanisms (Figure ): elastic (Thomson, coherent) scattering, Compton (incoherent)
bioRxiv (Cold Spring Harbor Laboratory), Jul 4, 2020
Protein aggregation is a widespread process leading to deleterious consequences in the organism, ... more Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, where the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced X-ray emission and X-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.
Using X-ray energies higher than those normally used for macromolecular X-ray crystallography (MX... more Using X-ray energies higher than those normally used for macromolecular X-ray crystallography (MX) at synchrotron sources can theoretically increase the achievable signal as a function of dose and reduce the rate of radiation damage . In practice, a major stumbling block to the use of higher X-ray energy has been the reduced quantum efficiency of silicon detectors as the X-ray energy increases, but hybrid photon counting CdTe detectors are optimised for higher X-ray energies, and their performance has been steadily improving. The potential advantages of using higher incident beam energy together with a CdTe detector for MX are explored, with a particular focus on the advantages that higher beam energies may have for MX experiments with microbeams or microcrystals. Our Monte Carlo calculations show a greater than a factor of 2 improvement in diffraction efficiency when using microbeams and microcrystals of 5 μm or less. These take into account the escape of photoelectrons from the crystal as well as entry from the surrounding material [3], both of which have now been incorporated into RADDOSE-3D.
Xylose isomerase (XI) is an industrially important metalloprotein studied for decades. Its reacti... more Xylose isomerase (XI) is an industrially important metalloprotein studied for decades. Its reaction mechanism has been postulated to involve movement of the catalytic metal cofactor to several different conformations. Here, a dose dependent approach was used to investigate the radiation damage effects on XI and their potential influence on the reaction mechanism interpreted from the X-ray derived structures. Radiation damage is still one of the major challenges of X-ray diffraction experiments and causes both global and site-specific damage. In this study, consecutive high-resolution data sets from a single XI crystal from the same wedge were collected and the progression of radiation damage was tracked over increasing dose (0.13-3.88 MGy). The catalytic metal and its surrounding amino acid environment experience a build-up of free radicals, and the results show radiation damage induced structural perturbations ranging from an absolute metal positional shift to specific residue motions in the active site. The metal shift is an artefact of global damage and the resulting unit cell expansion, but residue motion appears to be driven by the dose. Understanding and identifying radiation-induced damage is an important factor in accurately interpreting the biological conclusions being drawn.
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Papers by Elspeth Garman