No description available.

No description available.

Electron crystallography of two-dimensional crystals is a structure-determination method well suited to the study of membrane protein structure-function. Two-dimensional crystals consist of ordered arrays of protein within reconstituted lipid bilayers, an arrangement that mimics the natural membrane environment. In this work we describe our recent progress in the use of this method with three different proteins, each providing a window into a separate paradigm in the electron crystallographic pipeline. Specific crystallization conditions for human leukotriene C₄ synthase (LTC₄S) have previously been determined, but our continued refinement of purification and crystallization has identified a number of additional parameters that greatly affect crystal size and quality, and we have developed a protocol to rapidly and reproducibly grow large, non-mosaic crystals of LTC₄S. The human gamma-glutamyl carboxylase (GGCX) has also been crystallized, but is sensitive to cryo-EM sample preparation conditions and we present here the successful reproduction of crystallization and refinement of cryo-EM sample preparation conditions. Lastly, we describe our crystallization screens with the Vibrio cholerae sodium-pumping NADH:ubiquinone reductase complex (Na⁺-NQR), and identify the factors critical to membrane reconstitution of the complex, a necessary first step towards crystallization. We also describe a semi-quantitative crystal screening protocol we have developed that provides quick and accurate method to assess two- dimensional crystallization trials, and discuss some general observations in optimization of membrane protein purification and two-dimensional crystallization for electron crystallography.

No description available.

La membrane cellulaire est la barrière qui sépare l'intérieur des cellules de l'environnement extérieur. Elle se compose de lipides et de protéines. Les gènes codant pour les protéines membranaires représentent environ 30% des génomes. Les protéines membranaires sont synthétisées dans le cytosol par les ribosomes, mais suivent des voies spécifiques pour s'intégrer dans la membrane cellulaire. Les ribosomes en cours de traduction de protéines membranaires sont reconnus dans le cytosol et adressés à la membrane. Par la suite, les chaînes naissantes de protéines membranaires sont insérées dans la bicouche lipidique puis repliées de façons appropriées, ce mécanisme s'appelle la translocation. Le processus d'adressage est médiée par la particule de reconnaissance du signal (SRP) et son récepteur, tandis que la translocation est effectuée par un certain nombre de complexes de protéines membranaires.Cette thèse décrit deux des complexes impliqués dans cet adressage et translocation co-traductionnelle chez Escherichia coli : Le complexe ribosome-SRP-FtsY pour l'adressage en conformation «fermé» et le complexe dans lequel le ribosome est lié à l'holo-translocon (HTL) qui se compose de sept protéines membranaires. J'ai utilisé principalement la cryo-microscopie électronique pour caractériser ces complexes. La cryo-EM permet de déterminer la structure des échantillons biologiques à une résolution supérieure au nanomètre dans leur environnement natif, sans avoir à le cristalliser. Dans ce travail, j'ai bénéficié des améliorations récentes dans l'équipementet le traitement d'image.A partir d'un ensemble de données de cryo-EM obtenu par les membres du groupe, j‘ai déterminé la structure du complexe ribosome-SRP-FtsY en conformation «fermé» avec une résolution de 5.7 Å. Différentes stratégies de tri des calculsont été appliquées pour identifier la partie la plus homogène de l'ensemble des données. La structure montre un domaine bien résolu SRP ARN et SRP M avec une séquence signal liée. L'interaction entre les SRP et le ribosome pourrait être modélisée avec une grande fidélité. Cette structure révèle également que les GTPases SRP-ftsY sont détachées de la tétra-boucle de l'ARN et sont flexibles, libérant alors le site de sortie du ribosomepermettant la liaison du translocons.Dans le second projet, différentes approches ont été poursuivis pour résoudre la structure du complexe ribosome-HTL à haute résolution. Une structure initiale à 22 Å a été obtenue en mélangeant HTL solubilisés en détergent avec des ribosomes, démontrantla possibilité de préserver le complexe dans les conditions utilisées pour la préparation des grilles. J'ai ensuite exploré l'utilisation de nanodiscs et un nouveau détergent appelé LMNG pour stabiliser HTL dans des tampons sans détergent. Un deuxième ensemble de données a ensuite été recueilli à partir d'échantillon obtenu par gradient de fixation, la structure a été résolue à 17 Å. La préparation des échantillons a été optimisée utilisant entre autre les amphipoles. On a montré que deux types d'amphipole-HTL peuvent se lier au ribosome, et des structures de plus grande résolution devrait être obtenu à partir de ces échantillons. / The cell membrane is the barrier that separates the interior of cells from the outside environment. It consists of lipids and proteins. Genes encoding membrane proteins make up about 30% of the genome. Membrane proteins are synthesized in the cytosol by ribosomes, but employ special pathways to integrate into the cell membrane. Ribosomes translating membrane proteins are recognized by special factors in the cytosol and targeted to the membrane. Subsequently, nascent chains of the membrane proteins are inserted into the lipid bilayer and are folded into their proper structures, a process termed translocation. The targeting process is mediated by the signal recognition particle (SRP) and its receptor, while the translocation is performed by a number of membrane protein complexes.This thesis describes two of the complexes involved in co-translational targeting and translocation in Escherichia coli: The ribosome-SRP-FtsY targeting complex in the “closed” conformation and the complex of a ribosome with the holo-translocon (HTL) consisting of seven membrane proteins. I mainly used electron cryo-microscopy to characterize these complexes. Cryo-EM allows structural determination of biological samples at sub-nanometer resolution in their native environment, without the need to crystallize the specimen. In this work, I took advantage of the recent advances in both the hardware and the image processing.Starting from a cryo-EM dataset obtained by group members, I have determined the structure of ribosome-SRP-FtsY complex in the “closed” conformation at 5.7 Å resolution. Different computational sorting strategies were applied to identify the most homogeneous sub-pool of the dataset. The structure shows a well-resolved SRP RNA and SRP M domain with a signal sequence bound. The interaction between SRP and ribosome could be modeled with high confidence. This structure also reveals that the SRP-FtsY GTPases are detached from the RNA tetraloop and are flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.In the second project, different approaches were pursued to solve the structure of the ribosome-HTL complex at high resolution. An initial structure at 22 Å was obtained by mixing detergent-solubilized HTL with the ribosome, demonstrating that it is possible to preserve the complex under the conditions used for specimen preparation. I have then explored the use of nanodiscs and a new detergent called LMNG to stabilize HTL in detergent-free buffers. A second dataset was subsequently collected from a sample prepared by gradient-fixation, and the structure was solved at 17 Å. Sample preparation has been optimized further using amphipols. Two types of amphipol-HTL complexes were shown to bind to the ribosome, and higher resolution structures are expected to be obtained from these samples.

Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...

Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...