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Abstract: Crystallization is a key step in drug purification, offering low cost and facile scalability. The thesis investigates the role of heterogeneous nucleation templates in enhancing crystallization efficiency and controllability with a focus on biopharmaceutical applications, and examines the mechanisms of template-mediated nucleation, crystal growth, and morphology control using carbon-based templates, polymeric hydrogel templates, and microbial bio-templates. The interaction between inorganic salt and proteins was investigated and proteins themselves were also applied as the macromolecular templates. Carbon-based materials, including graphite and graphene oxide (GO), were investigated for their influence on lysozyme crystallization. Graphite reduced nucleation time by 57% compared to those without templates and demonstrated edge adsorption. GO exhibited a nonlinear effect, accelerating nucleation at low lysozyme concentrations (30 mg mL-1) while inhibiting it at higher concentrations (over 50 mg mL-1). Furthermore, a second strategy was pursued using heterogeneous templates based on poly (ethylene glycol) diacrylate (PEGDA) hydrogel microspheres (HMS). In contrast to the adsorption mechanism, the PEGDA HMS acts by releasing precipitant (0- 4 M NaCl) to create localized supersaturation gradients, thereby reducing nucleation time by 79%. Based on the mechanisms of templated crystallization observed in the lysozyme system, this work sought to explore the universality of these effects in inorganic systems critical to biomineralization and disease. The interaction between proteins and inorganic salts was further investigated in two model systems: lithium carbonate (Li'CO') and calcium oxalate (CaOx) with proteins (lysozyme, bovine haemoglobin and mRFP). In both systems, inorganic salt crystals serve as templates that influence subsequent protein adsorption and crystallization, leading to the formation of protein-salt composite crystal structures. Elevated salt concentrations consistently promoted nucleation kinetics. Proteins, however, exhibited complex effects: At low supersaturation, proteins like lysozyme inhibited Li'CO' nucleation by chelating Li'. Conversely, at high supersaturation, proteins self-assemble into oligomers or aggregates, providing additional nucleation sites and accelerating nucleation. In the CaOx system, lysozyme enhanced nucleation across its tested concentration range (0-70 mg mL-1). To bridge our findings on artificial templates to biological contexts, in vivo crystallization is further explored. Inspired by nature, the production of intracellular crystals in Bacillus thuringiensis (Bt) was studied, and its Cry1Ac gene was applied to form a crystal scaffold (CS) as the bio-template to generate crystal nanoparticles in Escherichia coli (E. coli). Through adaptive laboratory evolution (ALE) via serial passaging, we achieved a nearly tenfold increase in protein fluorescence level and produced biologically active nanocrystals with high solubility under alkaline conditions. By integrating heterogeneous nucleation theory with biomimetic strategies, our work elucidates diverse templating mechanisms, including surface, the creation of local supersaturation gradients, and inorganic salt templates. These understandings enable the rational design of templates to control crystallization outcomes. Furthermore, we establish a platform that applying the Cry gene from Bt as the crystal scaffold to function as bio-templates inside cells, demonstrating their potential in high-yield production of bioactive nanocrystals.

Open Access

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Abstract: Structure determination by X-ray diffraction is limited by crystal size and can be compromised by radiation damage when using very intense X-ray radiation. X-ray structure determination from partial diffraction data sets combined from multiple crystals is a potential solution, but its exploitation in chemistry and materials science is largely unrealized. Here we report the use of synchrotron radiation for multi-crystal X-ray diffraction (MCXRD) adapted for structure determination of metal-organic framework (MOF) materials with crystal dimensions too small for conventional single-crystal diffraction studies. We further show that radiation-induced chemical changes and degradation of diffraction quality can be alleviated. Our approach encompasses both rotation- and stationary-MCXRD measurements for 10 to 1000s of crystals with software-optimized combination of the multiple data sets. We report the crystal structures of six MOFs: MOF-919(Sc/Cu), MET-2, MIL-88B(Cr)-1,4-NDC, PCN-260(Sc), UiO-66, and UiO-66-MoO4 with unit cell dimensions ranging from 18−114 Å and crystal sizes from 0.5−480 µm3. This approach can address the challenges of structure determination in a regime of particle size and sample radiation sensitivity that lies between existing single-crystal X-ray diffraction and the emerging field of electron diffraction. MCXRD can provide accurate atomic-resolution structure determination for some of the most challenging cases in chemistry and materials science.

Open Access

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Abstract: X-ray diffraction (XRD) of microcrystals is signal-to-noise limited by the inherently weak diffraction. As such, Electron diffraction (ED) is increasingly used to measure diffraction data from submicron crystals, or those deemed too small for XRD due the stronger interaction of electrons with matter. However, many samples which are too thin for XRD are often too thick for ED using the currently available electron beam energies (<300 keV) and hence require thinning by focussed ion beam milling (FIB) which adds additional sample preparation steps. In addition to determining structures from nanocrystals, ED provides Coulomb potential data which are complementary to that obtained with XRD. As such ED data may be necessary to answer particular scientific questions. The macromolecular crystallography beamline, VMXm, at Diamond Light Source, has been optimised for maximising the S:N in XRD experiments with a variable focus high-energy (>20 KeV) X-ray beam, with in-vacuum endstation and the use of low background cryoTEM grids for crystal mounting [1], [2]. This has allowed VMXm to collect high-resolution rotation data from single crystals measuring ∼1.2 μm which were only previously tractable using an X-ray Free Electron Laser [3]. This has pushed the amenable sample envelope at synchrotrons to new dimensions and perhaps near to the practical limit of XRD. Indeed, simulations have predicted the limit to be ∼0.5 μm thick in the case of lysozyme, assuming photoelectron escape [4]. This XRD beamline opens up the possibilities to directly compare XRD and ED datasets and understand the complementarity of these experiments. In this work we present data from cubic human insulin crystals that have been thinned by FIB milling from ∼10 μm to various submicron thicknesses. 200 kV ED data were then collected from these lamellae before XRD data were measured from the same lamellae using VMXm. It was possible to obtain a complete XRD dataset to 2.45 Å using a 1.68 μm3 illuminated volume and a 2.04 Å ED dataset from the same 0.25 μm lamella. We have demonstrated that the data quality is comparable between ED and VMXm from the same crystal, while giving an opportunity to directly compare X-ray and electron derived maps. This includes the comparison of the radiation damage each experiment imparts on the sample [5] as well as the information content [6]. This work indicates that the usable sample envelope for synchrotron X-rays extends to much thinner samples than had been previously thought. It is also the first demonstration of ED and XRD measured from the same crystal volume enabling direct comparison of X-ray and electron derived data. Ultimately, the work will inform the design and use of high energy (MeV) ED instruments such as HeXI and how those can be complemented by XRD derived information from beamlines such as VMXm.

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Abstract: Due to radiation damage, the majority of metalloproteins structures are incorrect. Radiation damage in X-ray crystallography manifests itself either globally or at specific radiation sensitive sites. Global damage can be monitored from data processing statistics, whereas specific damage is more clandestine and presents as structural changes within the electron density. Serial crystallography using an X-ray free-electron laser promises a pseudo zero dose structure, however, the paucity of beamlines means beamtime is highly competitive. Method development, therefore, is required to collect low-dose structures using synchrotron X-ray crystallography. One dose-reducing phenomenon is photoelectron escape, where the generated photoelectrons escape the crystal volume before depositing their energy. This thesis conducted the first serial crystallography experiment at VMXm (Diamond Light Source, UK), where photoelectron escape is significant for the targeted microcrystal sizes. An oxidised iron intermediate in myoglobin, Compound II, was tested as FeIV-oxo “ferryl” intermediates, which are known to be particularly susceptible to radiation damage. Despite not being a formal heme peroxidase, myoglobin is an excellent model for testing dose-limiting techniques. An NADP+-specific glyceraldehyde 3’-phosphate dehydrogenase from the enteric pathogen Helicobacter pylori was also investigated. NADP+-specificity is unusual amongst GAPDHs and are therefore poised for therapeutic targets. The kinetics of GAPDHA were investigated, and amongst the first structures of an NADP+-specific GAPDH outside of photosynthetic organisms are reported. An underreported form of radiation damage was observed. Therefore, a transition to microcrystals for a prospective dose-series and time-resolved investigation was performed. A Mix and Quench Microcrystal Reactor was developed to initiate a reaction within microcrystals with rapid mixing and to trap intermediates by quenching in liquid ethane. Current systems exist for time-resolved crystallography or time-resolved cryoEM; however, a system was developed to react and spray microcrystals onto a TEM grid for use on the specific goniometry at VMXm.

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Abstract: The world is full of treasure troves of hoarded scientific samples - everything from pressed plants in herbaria and pinned insects in museum drawers to rocks from the Moon and asteroids in air-tight vaults. Generations of scientists have contributed to collecting, preserving and studying such specimens, amassing an immense amount of knowledge about our natural world and how it changes over time. As we develop ever more powerful technologies to explore them, these historical samples can lead us to new discoveries, as an international team of researchers recently found. Their work, published in Nature Communications, began with a 70-year-old viral sample tracked down in a low temperature freezer. This viral sample was of a Nudivirus that are economically important in agriculture, where they are used as biological agents to control some insect pests. However, they can be pests themselves, damaging large-scale crustacean and insect farming efforts. Using X-ray crystallography, the research team solved the lattice structure of a polyhedrin (occlusion body) from Nudiviridae, which adds to our knowledge of how viruses use protein self-assembly to protect themselves from the environment.