abstract: Over the last century, X-ray crystallography has been established as the most successful technique for unravelling the structure-function relationship in molecules. For integral membrane proteins, growing well-ordered large crystals is a challenge and hence, there is room for improving current methods of macromolecular crystallography and for exploring complimentary techniques. Since protein function is deeply associated with its structural dynamics, static position of atoms in a macromolecule are insufficient to unlock the mechanism. The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation. Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2018

abstract: This thesis focuses on serial crystallography studies with X-ray free electron lasers (XFEL) with a special emphasis on data analysis to investigate important processes in bioenergy conversion and medicinal applications. First, the work on photosynthesis focuses on time-resolved femtosecond crystallography studies of Photosystem II (PSII). The structural-dynamic studies of the water splitting reaction centering on PSII is a current hot topic of interest in the field, the goal of which is to capture snapshots of the structural changes during the Kok cycle. This thesis presents results from time-resolved serial femtosecond (fs) crystallography experiments (TR-SFX) where data sets are collected at room temperature from a stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an XFEL with the goal to obtain structural information from the transient state (S4) state of the cycle where the O=O bond is formed, and oxygen is released. The most current techniques available in SFX/TR-SFX to handle hundreds of millions of raw diffraction patterns are discussed, including selection of the best diffraction patterns, allowing for their indexing and further data processing. The results include two 4.0 Å resolution structures of the ground S1 state and triple excited S4 transient state. Second, this thesis reports on the first international XFEL user experiments in South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this new XFEL in a proof-of-principle experiment for the study of microcrystals of human taspase1 (an important cancer target) by SFX has been tested. The descriptions of experiments and discussions of specific data evaluation challenges of this project in light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å, are included in this report In summary, this thesis examines current techniques that are available in the SFX/TR-SFX domain to study crystal structures from microcrystals damage-free, with the future potential of making movies of biological processes. / Dissertation/Thesis / Masters Thesis Chemistry 2020

abstract: Novel electric field-assisted microfluidic platforms were developed to exploit unique migration phenomena, particle manipulation, and enhanced droplet control. The platforms can facilitate various analytical challenges such as size-based separations, and delivery of protein crystals for structural discovery with both high selectivity and sensitivity. The vast complexity of biological analytes requires efficient transport and fractionation approaches to understand variations of biomolecular processes and signatures. Size heterogeneity is one characteristic that is especially important to understand for sub-micron organelles such as mitochondria and lipid droplets. It is crucial to resolve populations of sub-cellular or diagnostically relevant bioparticles when these often cannot be resolved with traditional methods. Herein, novel microfluidic tools were developed for the unique migration mechanism capable of separating sub-micron sized bioparticles by size. This based on a deterministic ratchet effect in a symmetrical post array with dielectrophoresis (DEP) for the fast migration allowing separation of polystyrene beads, mitochondria, and liposomes in tens of seconds. This mechanism was further demonstrated using high throughput DEP-based ratchet devices for versatile, continuous sub-micron size particle separation with large sample volumes. Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) revolutionized protein structure determination. In SFX experiments, a majority of the continuously injected liquid crystal suspension is wasted due to the unique X-ray pulse structure of XFELs, requiring a large amount (up to grams) of crystal sample to determine a protein structure. To reduce the sample consumption in such experiments, 3D printed droplet-based microfluidic platforms were developed for the generation of aqueous droplets in an oil phase. The implemented droplet-based sample delivery method showed 60% less sample volume consumption compared to the continuous injection at the European XFEL. For the enhanced control of aqueous droplet generation, the device allowed dynamic triggering of droplets for further improvement in synchronization between droplets and the X-ray pulses. This innovative technique of triggering droplets can play a crucial role in saving protein crystals in future SFX experiments. The electric field-assisted unique migration and separation phenomena in microfluidic platforms will be the key solution for revolutionizing the field of organelle separation and structural analysis of proteins. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2020

abstract: Serial femtosecond crystallography (SFX) uses diffraction patterns from crystals delivered in a serial fashion to an X-Ray Free Electron Laser (XFEL) for structure determination. Typically, each diffraction pattern is a snapshot from a different crystal. SFX limits the effect of radiation damage and enables the use of nano/micro crystals for structure determination. However, analysis of SFX data is challenging since each snapshot is processed individually. Many photosystem II (PSII) dataset have been collected at XFELs, several of which are time-resolved (containing both dark and laser illuminated frames). Comparison of light and dark datasets requires understanding systematic errors that can be introduced during data analysis. This dissertation describes data analysis of PSII datasets with a focus on the effect of parameters on later results. The influence of the subset of data used in the analysis is also examined and several criteria are screened for their utility in creating better subsets of data. Subsets are compared with Bragg data analysis and continuous diffuse scattering data analysis. A new tool, DatView aids in the creation of subsets and visualization of statistics. DatView was developed to improve the loading speed to visualize statistics of large SFX datasets and simplify the creation of subsets based on the statistics. It combines the functionality of several existing visualization tools into a single interface, improving the exploratory power of the tool. In addition, it has comparison features that allow a pattern-by-pattern analysis of the effect of processing parameters. \emph{DatView} improves the efficiency of SFX data analysis by reducing loading time and providing novel visualization tools. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

abstract: Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on the order of hundreds of femtoseconds. However, currently available XFEL facility X-ray pulse structures waste the majority of continuously injected crystal sample, requiring a large quantity (up to grams) of crystal sample to solve a protein structure. Furthermore, mix-and-inject serial crystallography (MISC) at XFEL facilities requires fast mixing for short (millisecond) reaction time points (𝑡"), and current sample delivery methods have complex fabrication and assembly requirements. To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å. For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s. / Dissertation/Thesis / Supplementary Video D.1 - Droplet formation in a 3D printed droplet generator / Doctoral Dissertation Chemistry 2019

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20071号 / 医博第4164号 / 新制||医||1018(附属図書館) / 33187 / 京都大学大学院医学研究科医学専攻 / (主査)教授 黒田 知宏, 教授 松田 文彦, 教授 楠見 明弘 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

abstract: Scientists have used X-rays to study biological molecules for nearly a century. Now with the X-ray free electron laser (XFEL), new methods have been developed to advance structural biology. These new methods include serial femtosecond crystallography, single particle imaging, solution scattering, and time resolved techniques. The XFEL is characterized by high intensity pulses, which are only about 50 femtoseconds in duration. The intensity allows for scattering from microscopic particles, while the short pulses offer a way to outrun radiation damage. XFELs are powerful enough to obliterate most samples in a single pulse. While this allows for a “diffract and destroy” methodology, it also requires instrumentation that can position microscopic particles into the X-ray beam (which may also be microscopic), continuously renew the sample after each pulse, and maintain sample viability during data collection. Typically these experiments have used liquid microjets to continuously renew sample. The high flow rate associated with liquid microjets requires large amounts of sample, most of which runs to waste between pulses. An injector designed to stream a viscous gel-like material called lipidic cubic phase (LCP) was developed to address this problem. LCP, commonly used as a growth medium for membrane protein crystals, lends itself to low flow rate jetting and so reduces the amount of sample wasted significantly. This work discusses sample delivery and injection for XFEL experiments. It reviews the liquid microjet method extensively, and presents the LCP injector as a novel device for serial crystallography, including detailed protocols for the LCP injector and anti-settler operation. / Dissertation/Thesis / Doctoral Dissertation Physics 2015

Investigations of soft matter using ultrashort high intensity pulses have been made possible through the advent of X-ray free-electrons lasers. The last decade has seen the development of a new type of protein crystallography where femtosecond dynamics can be studied, and single particle imaging with atomic resolution is on the horizon. The pulses are so intense that any sample quickly turns into a plasma. This thesis studies the ultrafast transition from soft matter to warm dense matter, and the implications for structural determination of proteins.                    We use non-thermal plasma simulations to predict ultrafast structural and electron dynamics. Changes in atomic form factors due to the electronic state, and displacement as a function of temperature, are used to predict Bragg signal intensity in protein nanocrystals. The damage processes started by the pulse will gate the diffracted signal within the pulse duration, suggesting that long pulses are useful to study protein structure. This illustrates diffraction-before-destruction in crystallography. The effect from a varying temporal photon distribution within a pulse is also investigated. A well-defined initial front determines the quality of the diffracted signal. At lower intensities, the temporal shape of the X-ray pulse will affect the overall signal strength; at high intensities the signal level will be strongly dependent on the resolution. Water is routinely used to deliver biological samples into the X-ray beam. Structural dynamics in water exposed to intense X-rays were investigated with simulations and experiments. Using pulses of different duration, we found that non-thermal heating will affect the water structure on a time scale longer than 25 fs but shorter than 75 fs. Modeling suggests that a loss of long-range coordination of the solvation shells accounts for the observed decrease in scattering signal. The feasibility of using X-ray emission from plasma as an indicator for hits in serial diffraction experiments is studied. Specific line emission from sulfur at high X-ray energies is suitable for distinguishing spectral features from proteins, compared to emission from delivery liquids. We find that plasma emission continues long after the femtosecond pulse has ended, suggesting that spectrum-during-destruction could reveal information complementary to diffraction.

Photosystem I (PSI) aus dem thermophilen Cyanobakterium Thermosynechococcus elongatus ist ein transmembraner Protein-Pigment-Superkomplex der photosynthetischen Elektronentransportkette. Er wandelt die Energie des Lichts in elektrische Energie mit einer Quanteneffizienz von nahezu 100 % um. Dazu uberträgt PSI Elektronen von Plastocyanin bzw. Cytochrom c6 (Cyt c6) auf Ferredoxin. Die Struktur des PSI wurde bereits 2001 mit einer Auflösung von 2,5 Å beschrieben (Jordan et al. 2001). Es lässt sich zur Generierung von Photoströmen auf Elektrodenoberflächen assemblieren und zur Produktion von Biokraftstoffen mit Enzymen koppeln. Die elektrische Kontaktierung des PSI mit Elektrodenoberflächen kann durch Komplexierung mit dem mitochondrialem Cytochrom c aus Pferdeherz (Cyt cHH) erhöht werden. Aufgrund der Nutzbarkeit dieses Proteinkomplexes sollte geklärt werden, wie PSI und Cyt cHH wechselwirken und wie sich die Interaktion von der des nativen PSI-Cyt c6-Komplexes unterscheidet. Deshalb lag der Fokus meiner Arbeit darauf, die Bindung des Cyt c6 und seines Analogons Cyt cHH an PSI mit kinetischen, kalorimetrischen, theoretischen und strukturellen Methoden zu untersuchen. Das Cyt c6 bindet im reduzierten Zustand an PSI und verringert nach erfolgtem Elektronentransfer seine Affinität. Das Cyt cHH bindet dagegen sowohl im reduzierten als auch im oxidierten Zustand an PSI. Mit Hilfe der kinetischen Messungen habe ich Bedingungen identifiziert, unter denen PSI mit dem jeweiligen Cytochrom c einen stabilen Komplex eingeht. Mit Hilfe eines rigid-body dockings wurden potenzielle Bindungsstellen der beiden Cytochrome berechnet. Fur Cyt c6 ergab sich eine spezifische Bindungsstelle, die eine gute Übereinstimmung mit den von mir gemessenen Kinetiken sowie mit weiteren Literaturdaten zeigt. Diese Bindungsstelle korreliert mit der veröffentlichten Kostruktur des bakteriellen Reaktionszentrums mit Cyt c2 aus Rhodobacter sphaeroides. Demgegenüber sind mehrere Cyt cHH-Bindungsstellen ... / Photosystem I (PSI) from the thermophilic cyanobacterium Thermosynechococcus elongatus is a membrane-bound, multipigment protein supercomplex. It converts light to electrochemical energy with a quantum efficiency of almost 100 %. It reduces the luminal proteins plastocyanin and cytochrome c6 (Cyt c6) to oxidize the stromal protein Ferredoxin. The structure of PSI has been solved in 2001 at a resolution of 2,5 Å (Jordan et al. 2001). PSI can be assembled on an electrode surface to produce photocurrents and the generated electrons can be used for the production of biofuels. The mitochondrial cytochrome c from horse heart (Cyt cHH) binds strongly to both, PSI and the electrode surface, and can therefore be applied to improve the electrical coupling. Due to the practical use of the PSI-Cyt c complex, the aim of my thesis is to characterize the interaction of PSI with Cyt c6 and the analog Cyt cHH. To this end, the binding of both cytochromes to PSI was analyzed by kinetic, calorimetric, theory-based and structural methods. Cyt c6 binds to PSI while being reduced and decreases its affinity after transferring its electron. In contrast, Cyt cHH binds to PSI in both oxidation states, reduced and oxidized, with identical affinity. By means of kinetic measurements, I identified conditions in which PSI forms a stable complex with either of the two cytochromes. The positions of the cytochrome binding sites at PSI were calculated by a rigid-body docking. For the calculation with Cyt c6, the majority of the potential binding sites are located at the luminal side of PSI, close to P700. The theoretic properties of one of these binding sites are in good agreement with my own kinetic measurements and literature data. The position and orientation of Cyt c6 in this theoretic binding site is almost identical to the localization of Cyt c2 in cocrystals with the bacterial reaction center from Rhodobacter sphaeroides. The potential Cyt cHH binding sites are uniformly distributed over ...