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Abstract: A group of three deep-learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate), were created for analysis of micrographs of protein crystallization experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case and masks of crystallization droplets in addition to crystals in the second case, allowing the positions and sizes of these entities to be recorded. The creation of these tools used transfer learning, where weights from a pre-trained deep-learning network were used as a starting point and repurposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS, where they have the potential to absolve the need for any user input, both for monitoring crystallization experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug-discovery screening platform, also at DLS, to allow the automatic targeting of acoustic compound dispensing into crystallization droplets.

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Abstract: The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form β-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 Å resolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallo­graphic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing.

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Abstract: Cytochromes c'-α are nitric oxide (NO)-binding heme proteins derived from bacteria that can thrive in a wide range of temperature environments. Studies of mesophilic Alcaligenes xylosoxidans cytochrome c'-α (AxCP-α) have revealed an unusual NO-binding mechanism involving both heme faces, in which NO first binds to form a distal hexa-coordinate Fe(II)-NO (6cNO) intermediate and then displaces the proximal His to form a proximal penta-coordinate Fe(II)-NO (5cNO) final product. Here we characterize a thermally stable cytochrome c'-α from thermophilic Hydrogenophilus thermoluteolus (PhCP-α) to understand how protein thermal stability affects NO binding. Electron paramagnetic and resonance Raman spectroscopies reveal the formation of a PhCP-α 5cNO product, with time-resolved (stopped-flow) UV-visible absorbance indicating the involvement of a 6cNO intermediate. Relative to AxCP-α, the rates of 6cNO and 5cNO formation in PhCP-α are ∼11-fold and ∼13-fold lower, respectively. Notably, X-ray crystal structures of PhCP-α in the presence and absence of NO suggest that the sluggish formation of the proximal 5cNO product results from conformational rigidity: the Arg-132 residue (adjacent to the proximal His ligand) is held in place by a salt bridge between Arg-75 and Glu-135 (an interaction not present in AxCP-α or a psychrophilic counterpart). Overall, our data provide fresh insights into structural factors controlling NO binding in heme proteins, including 5cNO complexes relevant to eukaryotic NO sensors.

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Abstract: A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.

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Abstract: DNA triplexes, formed by the binding of a triplex-forming oligonucleotide (TFO) within the major groove of a duplex, have been shown to have potential in gene editing and DNA nanotechnology applications. Recently, metal complexes, including ruthenium polypyridyl intercalators, have been widely explored for their distinctive DNA recognition properties and ability to induce site-specific DNA cleavage. Structural information, showing how ruthenium complexes can interact with DNA triplexes, is required to aid the development of compounds capable of selectively targeting and stabilising triple helical structures. This thesis reports solution and crystal-phase characterisation of the binding of ruthenium polypyridyl complexes to DNA triplexes, including the first crystal structure of a complete triplex with intercalated Ru-dppz complexes. UV thermal denaturation experiments were used to assess triplex stability under various conditions related to those used for crystallisation. This included pH (4.0 to 8.0), different cations (Na+ , Mg2+, Ca2+, Sr2+) and spermine, all of which are known to influence triplex thermodynamic stability. The presence of Mg2+ increased the Tm of intermolecular triplexes by ~5 °C and intramolecular triplexes by approximately 10 °C, compared to in the absence of magnesium ions. The observed stability profiles provided valuable guidance for the selection of systems to take forward for crystallisation and structural analysis. The stability and binding preferences of both enantiomers of [Ru(phen)2(dppz)]2+ were then explored in solution by systematically extending the duplex component of a model triplex system. Spectroscopic analysis, including fluorescence spectroscopy and circular dichroism, revealed the -enantiomers bind to terminal CG and TA steps of the extended duplex. While the -enantiomer exhibited fluorescence emission consistent though all the extended systems, stabilisation of the triplex (with a Tm of +1.2 °C) was only observed with CG extensions, suggesting intercalation by the complex adjacent to the terminus of the TFO. Crystallisation of a unimolecular TFO led to the first high-resolution (2Å) X-ray crystal structure of a complete DNA triplex with intercalated ruthenium polypyridyl complexes. Two - [Ru(phen)2(dppz)]2+ complexes intercalated into the minor groove of the DNA triplex, adjacent to T-A:T triplets, separated by a Watson-Crick base pair. This violates the neighbour exclusion prin- ciple due to binding in adjacent DNA steps. Two -[Ru(phen)2(dppz)]2+ complexes also intercalated into TA/TA steps within a DNA duplex cross-over region between symmetry-related triplexes. Crystallisation screening, using the sequences studied in chapter 2, yielded additional crystal structures. A second structure, determined to near-atomic resolution (1.2 Å) revealed for the first time how Ru-dppz complexes can intercalate into the major groove of the underlying duplex, excluding the TFO from the crystal lattice. The intercalation of -[Ru(TAP)2(11-CN-dppz)]2+ in the TA/TA steps into the duplex major groove provides insight into the stacking requirements, as well as the dppz-moieties required to achieve major groove intercalation. Finally, a crystal structure resulting from the self-assembly of a G-rich TFO was obtained. This demonstrated that the TFO could assemble into a G-quadruplex in the presence of K+ . The crystal structure, determined to 1.15 Å resolution, featured G-tetrads, T-tetrads and a novel T:G octaplet motif, at the interface between two non-symmetry equivalent quadruplexes. Overall, these findings provide insights into the intercalation of ruthenium complexes within DNA triplexes, highlighting novel structure formation while emphasizing the importance of careful TFO and DNA triplex design for future studies.