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abstract: Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- Å wavelength, Bragg peaks were recorded to 8.5- Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- Å wavelength, and Bragg reflections were recorded to 3- Å resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins. / Dissertation/Thesis / Ph.D. Chemistry 2011

abstract: ABSTRACT X-Ray crystallography and NMR are two major ways of achieving atomic resolution of structure determination for macro biomolecules such as proteins. Recently, new developments of hard X-ray pulsed free electron laser XFEL opened up new possibilities to break the dilemma of radiation dose and spatial resolution in diffraction imaging by outrunning radiation damage with ultra high brightness femtosecond X-ray pulses, which is so short in time that the pulse terminates before atomic motion starts. A variety of experimental techniques for structure determination of macro biomolecules is now available including imaging of protein nanocrystals, single particles such as viruses, pump-probe experiments for time-resolved nanocrystallography, and snapshot wide- angle x-ray scattering (WAXS) from molecules in solution. However, due to the nature of the "diffract-then-destroy" process, each protein crystal would be destroyed once probed. Hence a new sample delivery system is required to replenish the target crystal at a high rate. In this dissertation, the sample delivery systems for the application of XFELs to biomolecular imaging will be discussed and the severe challenges related to the delivering of macroscopic protein crystal in a stable controllable way with minimum waste of sample and maximum hit rate will be tackled with several different development of injector designs and approaches. New developments of the sample delivery system such as liquid mixing jet also opens up new experimental methods which gives opportunities to study of the chemical dynamics in biomolecules in a molecular structural level. The design and characterization of the system will be discussed along with future possible developments and applications. Finally, LCP injector will be discussed which is critical for the success in various applications. / Dissertation/Thesis / Doctoral Dissertation Physics 2014

abstract: Photosystem II (PSII) is a large protein-cofactor complex. The first step in photosynthesis involves the harvesting of light energy from the sun by the antenna (made of pigments) of the PSII trans-membrane complex. The harvested excitation energy is transferred from the antenna complex to the reaction center of the PSII, which leads to a light-driven charge separation event, from water to plastoquinone. This phenomenal process has been producing the oxygen that maintains the oxygenic environment of our planet for the past 2.5 billion years. The oxygen molecule formation involves the light-driven extraction of 4 electrons and protons from two water molecules through a multistep reaction, in which the Oxygen Evolving Center (OEC) of PSII cycles through 5 different oxidation states, S0 to S4. Unraveling the water-splitting mechanism remains as a grant challenge in the field of photosynthesis research. This requires the development of an entirely new capability, the ability to produce molecular movies. This dissertation advances a novel technique, Serial Femtosecond X-ray crystallography (SFX), into a new realm whereby such time-resolved molecular movies may be attained. The ultimate goal is to make a “molecular movie” that reveals the dynamics of the water splitting mechanism using time-resolved SFX (TRSFX) experiments and the uniquely enabling features of X-ray Free-Electron Laser (XFEL) for the study of biological processes. This thesis presents the development of SFX techniques, including development of new methods to analyze millions of diffraction patterns (~100 terabytes of data per XFEL experiment) with the goal of solving the X-ray structures in different transition states. ii The research comprises significant advancements to XFEL software packages (e.g., Cheetah and CrystFEL). Initially these programs could evaluate only 8-10% of all the data acquired successfully. This research demonstrates that with manual optimizations, the evaluation success rate was enhanced to 40-50%. These improvements have enabled TR-SFX, for the first time, to examine the double excited state (S3) of PSII at 5.5-Å. This breakthrough demonstrated the first indication of conformational changes between the ground (S1) and the double-excited (S3) states, a result fully consistent with theoretical predictions. The power of the TR-SFX technique was further demonstrated with proof-of principle experiments on Photoactive Yellow Protein (PYP) micro-crystals that high temporal (10-ns) and spatial (1.5-Å) resolution structures could be achieved. In summary, this dissertation research heralds the development of the TR-SFX technique, protocols, and associated data analysis methods that will usher into practice a new era in structural biology for the recording of ‘molecular movies’ of any biomolecular process. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015

abstract: Time-resolved serial femtosecond crystallography is an emerging method that allows for structural discovery to be performed on biomacromolecules during their dynamic trajectory through a reaction pathway after activation. This is performed by triggering a reaction on an ensemble of molecules in nano- or microcrystals and then using femtosecond X-ray laser pulses produced by an X-ray free electron laser to collect near-instantaneous data on the crystal. A full data set can be collected by merging a sufficient number of these patterns together and multiple data sets can be collected at different points along the reaction pathway by manipulating the delay time between reaction initiation and the probing X-rays. In this way, these ‘snapshot’ structures can be viewed in series to make a molecular movie, allowing for atomic visualization of a molecule in action and, thereby, a structural basis for the mechanism and function of a given biomacromolecule. This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2018

abstract: G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data. Additionally, differential and specific cholesterol binding in the CCK receptor subfamily was observed while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. Mutation of this conserved CRAC sequence of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase, however, as most techniques, it has limitations. Using an optimized SFX experimental setup in a helium atmosphere we determined the room temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution and compared it with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, we demonstrated the capability of utilizing high XFEL beam transmissions, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete data set. The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

京都大学 / 新制・課程博士 / 博士(理学) / 甲第22990号 / 理博第4667号 / 新制||理||1670(附属図書館) / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)教授 山本 潤, 教授 石田 憲二, 教授 田中 耕一郎 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

We have built a spectrometer to investigate the Near-Infrared (NIR) range of this radiation, which is used for bunch diagnostics at the European X-ray Free-Electron Laser. This could give information on the development of microbunching, periodic features in the longitudinal charge profile of the bunches which have a negative impact on the operation of the facility. In general it offers an ability to investigate the influences of the laser heater, the compression, and other factors that affect the structure of the bunches. The CDR is generated 1934 m after the injector, at full acceleration. The spectrometer is based around the KALYPSO detector system, able to read out from a 256 pixel linear array detector at MHz frequencies, making it possible to obtain single bunch readings during current user operation of the facility, at 1.1 MHz. KALYPSO has an InGaAs sensor, sensitive in the range 0.9 – 1.7 μm. A 40 mm N-SF11 equilateral prism is used for dispersion. First measurements have been taken, and CDR has been detected. The spectrometer needs further calibration and resolution was lacking, but it can offer insight in to relative changes, and bunch-to and can be used as for fingerprinting the beam. A reduction in signal in the sensitive range and a skew towards longer wavelengths was seen when going from uncompressed to compressed beam. When varying the power of the laser heater the behavior varied from run to run, with changing machine settings. In some cases the CDR was attenuated, while FEL intensity initially increased, until the induced energy spread from the laser heater was large enough to inhibit the FEL process. Another, less expected, behaviour was also observed, where the initially low CDR intensity at first increased, while FEL intensity stayed the same, before it then followed the same pattern as in the first case.

Ce travail de thèse suit l'apparition récente de ces nouvelles sources intenses et courtes de rayonnement dans la gamme X/XUV que sont les lasers X/XUV à électrons libres (XFEL). Contrairement aux sources optiques qui déposent principalement leur énergie via les électrons libres, les photons X/XUV déposent leur énergie dans la matière par la photoionisation de couches internes avec éjection de photo-électrons, suivie par l'éjection d'électrons Auger et d'électrons de recombinaison à trois corps dans la distribution d'électrons libres. Le chauffage se fait donc par l'intermédiaire de la structure atomique. La forte intensité des XFELs permet de faire jusqu'à un trou par atome dans un solide produisant ainsi, sur une échelle femtoseconde, un état exotique fortement hors-équilibre appelé solide creux. Cet état exotique instable se désexcite via un ensemble de processus atomiques élémentaires. Nous nous sommes intéressés dans cette thèse au développement d'outils permettant de calculer la cinétique des populations atomiques, couplée à la cinétique des électrons libres, pendant la transition à densité ionique constante, de solide à plasma dense en passant par l'état de solide creux, induit par le rayonnement XFEL irradiant une cible solide. Tout le défi ici a été de calculer cette cinétique couplée hors-équilibre entre ces états de la matière de nature très différente. Pour répondre a ce défi nous avons développé deux modèles cinétiques d'interaction XFELsolide, pour lesquels la description d'un solide comme un plasma froid dégénéré nous a permis d'utiliser une même approche plasma pendant l'ensemble de la transition du solide au plasma. L'ensemble de la physique atomique HETL d'intérêt ayant lieu à densité du solide, bien avant la détente de la matière, nous avons développé deux codes associés à ces modèles pour une utilisation à densité ionique constante. Pour aborder l'étude nous nous sommes d'abord concentrés sur la cinétique des électrons liés en supposant une distribution d'électrons libres à l'équilibre (ce qui suppose une thermalisation instantanée des électrons libres). Dans le cadre de l'approche de plasma dense étendue jusqu'au solide, nous avons développé un modèle collisionnel-radiatif généralisé. Cette généralisation passe par l'identification d'un lien entre état solide et plasma au niveau des processus atomiques élémentaires. Le code développé à partir de ce modèle nous a permis d'étudier des résultats expérimentaux et ainsi d'améliorer notre description des effets de densités dans les plasmas denses. Dans une seconde partie nous avons ajouté à l'étude la cinétique des électrons libres en considérant une distribution d'électrons libres hors-équilibre. Le code associé, basé sur la discrétisation de cette distribution et son couplage avec les états liés, nous a permis d'étudier le rôle des processus atomiques élémentaires dans la thermalisation de la distribution d'électrons libres. / This work follows the recent development of the free electron lasers in the X-ray and XUV-ray range (XFEL). Unlike optical sources that deposit their energy via the free electrons, the X/XUV photons deposit their energy directly via photoionization of inner shell electrons with the ejection of photo-electrons, followed by the ejection of Auger electrons and three body recombination electrons in the free electron distribution. The matter is thus heated via the atomic structure. The high XFEL intensity allows one to make up to one hole per atom in a solid, thus producing, on a femtosecond time scale, an exotic state, highly out of equilibrium, called hollow cristal. This unstable exotic state deexcite via a set of elementary atomic processes. In this work we were interested in the development of tools to calculate the atomic population kinetics, coupled to the free electron kinetics, during the transition at constant ionic density, from solid to dense plasma, induced by an XFEL irradiating a solid target. The goal here was to calculate this out of equilibrium coupled kinetics between states of matter having a very different nature. To address this problem we have developed two kinetics models of XFEL interaction with solids. In both these models the description of the solid as a cold degenerated plasma allowed us to use the same plasma approach during all the solid-plasma transition. Considering the fact that all the atomic physics takes place at solid density, way before the matter relaxation, we have developed two codes, associated with these two models, for a use at constant ionic density. To approach this study, we first focused on the bound electron kinetics assuming a free electron distribution at equilibrium (i.e. hypothesis of instantaneous thermalization of the free electrons). In the framework of the dense plasma approach extended up to the solid state, we have developed a generalized collisional radiative model. This generalization goes through the identification of a link between the solid state and the plasma state for the elementary atomic processes. The code associated with this model allowed us to study experimental results and to improve our description of the density effects in dense plasmas. In a second part the free electron kinetics is included in the model with a free electron distribution out of thermodynamic equilibrium. The associated code, based on the discretization of this distribution and its coupling with bound atomic states allowed us to study the role of the atomic elementary processes in the free electron distribution thermalization.

Bei der lichtinduzierten Oxidation von Wasser im Photosystem II (PSII) werden zwei wassermoleküle im katalytischen Zyklus des Metallclusters (Mn4CaO5) benötigt, und vier Protonen aus dem Cluster in den Lumen abgegeben. Daher ist es für das Verständnis des Mechanismus´ der Wasseroxidation von entscheidender Bedeutung, die Veränderung der Protonierungszustände am cluster während der Katalyse zu untersuchen. Hierbei sollten sowohl die Wasserkanäle für die Zuführung der Substratwassermoleküle als auch die Transportwege für die Freisetzung der Protonen untersucht werden. Deshalb wurde in meiner ersten Veröffentlichung ein neues Protokoll entwickelt, um einzelne große Kristalle von dPSII mit einer Länge von ~3 mm in der Längsachse zu züchten. Diese Kristalle mit einer Auflösung von ca. 8 Å gemessen. Um eine höhere Auflösung zu erzielen, ist die Verbesserung der Kristallqualität essenziell. Daher wurde in meiner zweiten Veröffentlichung die Struktur des Detergens-Protein-Komplexes von dPSII mit βDM, durch Anwendung von SANS in Kombination mit SAXS untersucht. Die Ergebnisse zeigten, dass βDM eine monomolekulare Schicht um dPSII bildet. Darüber hinaus konnten freie Mizellen von βDM in der Lösung nachgewiesen werden. Damit ist eine weitere Optimierung der βDM-Konzentration in der Proteinlösung erforderlich, um die Bildung von freien Mizellen zu minimieren. In meiner dritten Veröffentlichung wurde die strukturelle Dynamik in den Wasserkanälen, während des S2-S3 Übergangs mit Hilfe der XFEL untersucht. Ein Datensatz mit einer hohen Auflösung von 1,89 Å wurde durch die Zusammenführung von Daten gewonnen, die während des S2-S3 Übergangs gesammelt wurden. In Anbetracht der Analyse der zusammengeführten Daten und der einzelnen Zeitpunkte, die während des S2-S3 Übergangs gesammelt wurden, ist es wahrscheinlich, dass ein Substratwasser durch den O1-Kanal geliefert wird. Im Gegensatz dazu wird ein Proton aus dem Cluster durch den Cl1 Transportweg in Richtung Lumen freigesetzt. / The light-induced oxidation of water in Photosystem II (PSII) requires incorporating two water molecules in the catalytic cycle of the active metal cluster (Mn4CaO5). Furthermore, four protons are released towards the bulk from the cluster. Therefore, tracking the change of protonation states at the active catalytic site and the surrounding protein side chains during catalysis and elucidating the pathways of water substrate insertion and proton release are crucial to understanding the water oxidation mechanism. Therefore, in the first study of my work, a new protocol was developed to grow single large dPSIIcc crystals with a length of ~3 mm in the long axis. These crystals, soaked in D2O containing buffer, diffracted to about 8 Å resolution. Improving the crystal quality is crucial for achieving a better resolution. Consequently, in the second study of my work, the structure of the detergent-protein complex of βDM-dPSIIcc has been investigated by applying SANS in combination with SAXS. The results showed that βDM is forming a monomolecular layer around the dimeric PSII core complex (dPSIIcc). Moreover, the SAXS data detected a peak assigned to the free micelles of βDM. These results raise the necessity to optimize the βDM concentration in the protein solution to avoid the possible excess of free micelles. In the third study of my work, the structural dynamics in the water channels connecting the cluster to the lumen during the S2  S3 transition were investigated using serial femtosecond XFEL. A high-resolution data set was obtained at a resolution of 1.89 Å by combining data collected at RT. Considering the analysis of the combined data and the individual time points collected during the S2  S3 transition, it is likely that the substrate water insertion into the open coordination site of the Mn1 ion is delivered through the O1 channel. In contrast, a proton from the cluster is released towards the bulk through the Cl1 A channel.