Crystallography is from far the most contributing technique for the structure analysis of macromolecules at atomic resolution. In this thesis, instrumentation development issues to improve and accelerate experimental procedures for X-ray diffraction experiments are tackled. Indeed the preparation steps of protein crystals for X-ray diffraction data collection are the main causes of forming a bottleneck towards automated pipelines from protein crystallization to structure resolution. Firstly, an emerging method in today macromolecular crystallography is the room temperature in situ X-ray diffraction of protein crystal samples in their crystallization drops, with proven benefits in crystal screening and also structure resolution. However, it requires a great number of crystals to be centered and diffracted in a row. Thus a fully automated system providing a solution to this requirement is presented and assessed in this manuscript as one of the results of this PhD studies. Secondly, in this manuscript, studies and developments on automating harvesting, cryo-protecting and flash-cooling steps of protein crystals preparation for X-ray diffraction are reported, as well as assessment experiments and results. With a new robotic approach, crystals are manipulated with a micro-gripper on a 6-axis robotic arm to prepare and to analyze crystals with 360° rotation possibility for cryo-temperature single wavelength X-ray diffraction. Lysozyme and NikA Fe-EDTA protein crystals has been prepared and diffracted with this new method. Structural comparisons show no differences between the new methodology and the manual one, while robustness, repeatability and experimental time are significantly improved. At last, different integration scenarios of the presented methodologies, highlights their interest in fully automated macromolecular crystallography pipelines. / La cristallographie est la technique qui contribue le plus à l'analyse des structures des macromolécules biologiques à la résolution atomique. Dans ce manuscrit de thèse nous abordons des développements instrumentaux pour l'amélioration et l'accélération des étapes expérimentales dans la procédure de mesure de la diffraction aux rayons X. En effet, les étapes de préparation des cristaux de protéine à la diffraction aux rayons X constituent la cause principale du goulot d'étranglement dans les plateformes à haut débit de la cristallisation des protéines jusqu'à la résolution des structures. Premièrement, la diffraction in situ aux rayons X des cristaux à la température ambiante, dans les plaques de cristallisation, est une méthodologie émergeante dans la cristallographie des protéines avec des capacités bénéfiques dans le criblage des cristaux mais aussi dans la résolution de structures. Cependant, un grand nombre de cristaux devront être centrés puis analysés par la diffraction aux rayons X automatiquement l'un à la suite de l'autre. Ainsi, un système automatisé répondant à cette exigence est présenté et évalué dans ce manuscrit comme étant l'un des résultats des études menées au cours de cette thèse. Deuxièmement, des études et des développements d'automatisation des étapes d'extraction et de micromanipulation, de cryo-protection et de congélation rapide pour la préparation des cristaux à la diffraction aux rayons X sont décrits dans ce manuscrit, ainsi que les résultats des expériences et des évaluations. Avec une approche nouvelle, les cristaux sont manipulés grâce à une micro-pince montée sur un bras robotique 6-axes pour les préparer et les analyser avec la possibilité de rotation de 360° pour la diffraction aux rayons X à longueur d'onde constate et à température cryogénique. Des cristaux des protéines lysozyme et NikA Fe-EDTA ont été préparés et diffractés avec cette nouvelle méthode. La comparaison structurale ne montre pas de différence entre la nouvelle méthode et celle manuelle, cependant la robustesse, la répétabilité et le gain de temps d'expériences sont significativement améliorés. Finalement, différents scénarios d'intégration des méthodologies présentées, met en évidence leurs intérêts dans les plateformes tout automatisés de cristallographie des macromolécules biologiques.

The high resolution structure of protein molecules and protein-protein complexes is important to investigate their functions. Today, large 3D or 2D crystals are required to obtain protein structures by X-ray crystallography or conventional Electron Microscopy, respectively. However, production of such crystals of good quality is a solely empirical procedure, which relies on screening numerous crystallization conditions. At the same time, multilayered protein crystals are often a by-product of attempts to grow 3D or 2D crystals and could be obtained more easily. So far, multilayered protein crystals have not been used in electron microscopy for structure determination, as the interpretation of an electron diffraction pattern is rather complicated. In this thesis we present the first protein structure bovine liver catalase at 4 Å resolution solved using electron diffraction data from multilayered crystals. 55 diffraction patterns (17 tilt series) were recorded and used for the reconstruction. The tilt geometry of each individual diffraction pattern was determined by a least-squares algorithm or Laue zone analysis to perform spot indexing. The phase problem was solved by molecular replacement. The influence of the missing data cone on the self-rotation function and interpretation of reconstructed map is discussed.

Acoustic focusing of microscale protein crystals with acoustophoresis technology could reduce clogs during experiments with the scientific technique serial femtosecond x-ray crystallography (SFX). SFX determines molecular structures of proteins, these structures are valuable in drug discovery and fundamental biomedical research. Lysozyme crystals were focused in their own mother liquor and dilutions with PBS buffer. The aim of these tests were to study how the acoustic contrast factor Φ changes with the medium. Recorded experiments were analyzed using the particle tracking software Trackmate to extract velocities and radii. The lysozyme crystals changed morphologies in large dilutions of PBS buffert, they either became rounder or broke into fragments. The changed forms are likely caused by dissolution behaviors; some dilutions were unstable, but not unstable enough to dissolve the crystals completely.  Measured velocities during focusing of the crystals had large variance. Sinusoidal fits of the velocities had significant increases in amplitudes for larger dilutions of PBS. A change in acoustic contrast factor Φ could be the cause for the increased amplitudes, but the results do not rule out other causes. There are currently major knowledge gaps about using protein crystals as particles with acoustophoresis technologies, hence many ideas for future works have been proposed in this master thesis report. / Akustisk fokusering av mikrometers-stora proteinkristaller med hjälp av ultraljudsteknik skulle kunna reducera proppar under experiment med tekniken seriell femtosekundskristallografi (SFX). SFX kan avgöra vilken struktur proteinmolekyler har, dessa strukturer är värdefulla för industriell utveckling av nya läkemedel och fundamental biomedicinsk forskning. Proteinkristaller av lysozym har fokuserats i sin egen kristalliseringslösning och utspädningar av PBS. Målet med de här experimenten var att se om den akustiska flödeskontrasten Φ kunde bli påverkad. Fokuseringar spelades in och partiklarnas hastigheter vid fokuseringarna mättes med hjälp av Trackmate (en mjukvara för partikelspårning). I de större utspädningarna förändrades lysozymkristallernas former, de blev antingen mer runda eller så blev de små bitar. Förändringen skedde förmodligen på grund av upplösningsmekanismer i instabila utspädningar.  Uppmätta hastigheter vid testerna hade stor varians, men kurvanpassningar av data tyder på att hastigheterna ökar signifikant med utspädningar av PBS. Den akustiska flödeskontrasten Φ kan vara orsaken för ökade hastigheter, men andra hypoteser gick inte att utesluta med resultaten. Stora kunskapshål finns om akustisk fokusering av proteinkristaller, därför har många idéer för framtida experiment och arbeten föreslagits i rapporten.