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Abstract: The increasing availability of ultrabright Light Sources is facilitating the study of smaller crystals at faster timescales but with an increased risk of severe X-ray damage, leading to developments in multi-crystal methods such as serial crystallography (SX). SX studies on crystals with small unit cells are challenging as very few reflections are recorded in a single data image, making it difficult to determine the orientation matrix for each crystal and thus preventing the combination of the data from all crystals for structure solution. We herein present a Small-Rotative Fixed-Target Serial Synchrotron Crystallography (SR-FT-SSX) methodology, in which rotation of the serial target through a small diffraction angle at each crystal delivers high-quality data, facilitating ab initio unit cell determination and atomic-scale structure solution. The method is benchmarked using microcrystals of the small-molecule photoswitch sodium nitroprusside dihydrate, obtaining complete data to dmin = 0.6 Å by combining just 66 partial datasets selected against rigorous quality criteria.

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Abstract: X-ray crystallography is an important biological research technique that has provided most of the structures in the Protein Data Bank. An inherent challenge of crystallography is that the X-rays which produce diffraction images also cause damage to the crystal, resulting in loss of diffraction quality as the absorbed dose increases. To minimise this effect, serial crystallography techniques have been developed to spread dose over many crystals, by collecting a small amount of data, usually only one diffraction image, from each. This is especially beneficial for metalloproteins since metal centres are particularly susceptible to site-specific radiation damage and are important to the function of many proteins. The benefits of serial crystallography include the ability to perform experiments at room temperature, where proteins are more likely to be in physiologically relevant conformations, rather than at the cryogenic temperatures usually used to limit radiation damage. Room temperature also allows increased diffusion and molecular motions, so reactions can take place within protein crystals. These can be investigated with time-resolved serial experiments, capturing structures of short-lived intermediate states of a protein undergoing a dynamic process. An important part of any time-resolved experiment is reaction initiation, including ensuring the reaction begins in the whole sample at the same time. This thesis describes the development of a method for time-resolved serial crystallography using the fixed-target chip system available on beamline I24 at Diamond Light Source, which is also applicable to serial femtosecond crystallography at X-ray free electron laser (XFEL) sources. These experiments explore the structures of highly radiation-sensitive metalloproteins, including time-resolved studies of nitric oxide binding in the haem site using a UV laser pump-probe technique and a photocaged substrate molecule.

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Abstract: The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump–probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

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Abstract: Macromolecular X-ray crystallography is the predominant method used for protein structure determination; however, data collection is impeded by sample radiation damage. Global radiation damage manifests as a loss of diffraction intensity, whereas specific radiation damage causes structural changes at radiation sensitive features. This thesis investigates the radiation sensitive, iron binding protein FutA from the cyanobacteria Prochlorococcus MED4. FutA is thought to function primarily as a periplasmic ferric iron (Fe3+) binding protein but may facilitate a second function as an intracellular ferrous iron (Fe2+) binding protein. Structure determination of FutA by X-ray crystallographic methods is impeded by X-ray induced photoreduction of the ferric iron to ferrous iron. Thus, radiation dose-limiting data collection techniques are required to determine an accurate structure of the ferric state. Many radiation dose-limiting data collection techniques have strict sample requirements that specify crystal size, number, and morphology. This thesis discusses the construction of a crystallisation phase diagram for FutA to elucidate the relationship between crystallisation components and the resulting crystal sample. The information provided by the phase diagram allowed suitable FutA samples to be produced for radiation dose-limiting structure determination. As a result, this thesis presents the first radiation-damage-free structure of ferric bound FutA solved using serial femtosecond crystallography (SFX). The sensitivity of FutA to X-ray induced photoreduction was utilised to investigate the potential dual ferric and ferrous iron binding function of FutA. The structural response to photoreduction was determined using dose-series produced by serial synchrotron crystallography (SSX) and was corroborated by the capture of the ferrous state using lowdose, single-crystal X-ray diffraction with a home-source. The photoreduction of FutA was characterised by a repositioning of Arg203 which is thought to stabilise the change in the iron redox state. The ability of FutA to stabilise the change in the redox state from ferric to ferrous iron may be indicative of an in vivo ferrous iron binding function. Many of the radiation-limiting data collection techniques used for FutA structure determination required large amounts of protein material. As a result, these techniques are unsuitable for proteins where the production of crystals is difficult. VMXm is a new micro/nano-focus beamline at Diamond Light Source (UK) which aims to facilitate data collection from crystals limited in size and number. At crystal sizes targeted by VMXm, improvements in crystal lifetime are expected, because global radiation damage is reduced due to the effect of photoelectron escape. In this thesis, the effect of photoelectron escape on the lifetime of microcrystals (3 – 11 µm) was investigated, where crystal lifetime was found to improve by a factor of 1.4 between a 3 µm and 10 µm crystal at 22.3 keV X-ray energy.

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Abstract: Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.