No description available.

<p>The ribosome is part of the indispensable machinery of every living cell. This large macromolecule, which decodes messenger RNA to produce proteins, is the subject of intense study as the mediator of an essential process. The prokaryotic ribosome is a major target for antimicrobial therapy, as its structure differs significantly from the eukaryotic ribosome. At present, the in vivo process of translation on the mature bacterial, or 70S, ribosome is well studied and increasingly understood, while the process of assembling the small (30S) and large (50S) subunits of this complex ribonucleoprotein enzyme has mostly been studied in vitro. Consequently, the significance of in vivo events such as ribosomal RNA (rRNA) maturation and factor-mediated maturation is incompletely understood. By studying the nature and structure of an in vivo assembled immature 30S subunit, this thesis aims to gain a better understanding of the key events in 30S subunit biogenesis. Deletion of the assembly cofactor Ribosome Maturation Factor M (RimM) results in slow growth, inefficient rRNA processing, and accumulation of nonfunctional, immature 30S subunits. This work presents the first cryo-EM model of the immature 30S purified from a RimM knockout strain of <em>E. coli</em>. The structure reveals distortion of the decoding centre and a disrupted 50S-binding interface, attesting to the importance of rRNA processing in 30S maturation. Additionally, the model suggests consequences for ribosomal protein incorporation and rRNA domain position relative to the mature 30S.</p> / Master of Science (MSc)

Macromolecular assemblies such as the ribosome, spliceosome, polymerases are imperative for cellular functions. The current understanding of these important machineries and many other assemblies at the molecular level is poor. The lack of structural data for many macromolecular assemblies further causes a bottleneck in understanding the cellular processes and the various disease manifestations. Hence, it is essential to characterize the structures and molecular architectures of these macromolecular assemblies. Though the number of 3-D structures for individual proteins structures or domains in the Protein Data Bank (PDB) is growing, the number of structures deposited for macromolecular assemblies is relatively poor. Hence, apart from the use of experimental techniques for characterizing macromolecular assembly structures, the use of computational techniques would help in supplementing the growth of macromolecular assembly structures. This thesis deals with the use of integrative approaches where computational methods are combined with experimental data to model and understand the mechanistic features of macromolecular assemblies with a special focus on a sub-complex of the spliceosome machinery. Chapter 1 of this thesis provides an introduction to protein-protein interactions and macromolecular assemblies. Further, the modelling of macromolecular assemblies using integrative methods are discussed, with a subsequent introduction to the spliceosome machinery. In chapter 2, modelling studies were performed on the proteins involved in the general amino acid control mechanism, which is triggered in yeast under amino acid starvation conditions. The proteins involved in the study were Gcn1, a ribosome binding protein and the RWD-domain containing proteins Gcn2, Yih1, Gir2 and Mtc5. From laboratory experiments it is known that in order for Gcn2 activation, an eIF2α kinase, its RWD-domain has to bind to Gcn1 and the residue Arg-2259 is important for this interaction. As the 3-D structure for the Gcn1 region containing Arg-2259 is not currently available, its 3-D structure was inferred using fold recognition and comparative modelling techniques. Further, in order to understand the Gcn2 RWD domain-Gcn1 molecular interaction, a complex structure was inferred by using a restrained protein-protein docking procedure. As the proteins, Yih1 and Gir2 are known to bind to Gcn1 using their RWD-domains, first the structures of the RWD-domain containing proteins including Mtc5 were inferred using a Gcn2 RWD domain NMR structure. Additionally, the Gcn1-Gcn2 complex was used to build a set of complexes to explain the binding of other RWD domain containing proteins Yih1, Gir2 and Mtc5. The important molecular interactions were obtained on analysing the interacting residues in these complexes. Thus, the Gcn1-Gcn2 interaction at the molecular level has been proposed for the first time. Future experiments guided by the protein-protein complex models and the proposed set of mutations should provide an understanding about the critical molecular interactions involved in the general amino acid control mechanism. Chapter 3 describes an integrative approach that was used to decipher a pseudo-atomic model of the closed form of human SF3b complex. SF3b is a multi-protein complex containing seven components – p14, SF3b49, SF3b155, SF3b145, SF3b130, SF3b14b and SF3b10. It recognizes the branch point adenosine in the pre-mRNA as part of U2 snRNP or U11/U12 di-snRNP in the spliceosome. Although, the cryo-EM map for human SF3b complex has been available for more than a decade, the structure and relative spatial arrangement of all components in the complex are not yet known. The integrative modelling approach used here involved utilizing structural data in the form of available X-ray and NMR structures, fold recognition and comparative modelling as well as currently available experimental datasets, along with the available cryo-EM density map to provide a model with high structural coverage. Hence, the molecular architecture of closed form human SF3b complex was derived that can now provide insights into the functioning of SF3b in splicing. This might also help the future high resolution structure determination efforts of the entire human spliceosome machinery In chapter 4, the molecular architecture of the closed form of SF3b complex obtained from the use of integrative modelling approach (Chapter 3) is extensively discussed. The structure-function relationships for some of the SF3b components based on the pseudo-atomic model has also been provided. In addition, the extreme flexibility associated with some of the SF3b components based on dynamics analysis has also been examined. Further, using an existing U11/U12 di-snRNP cryo-EM map and the closed form SF3b complex pseudo-atomic model, an open form of the SF3b complex was modelled and the component structures were fit into it. Hence, it was found that the transition between closed and open forms is primarily caused by a flap containing the HEAT repeat protein, SF3b155. This Protein is also known to harbour cancer causing mutations and has the potential to affect the Closed to open transition as well as SF3b complex structure and stability. Thus, this provides a framework for the future understanding of the closed to open transition in SF3b functioning within the spliceosome. Chapter 5 builds upon the integrative modelling approach (Chapter 3) that proposed the molecular architecture of the closed form of human SF3b complex and an open form of SF3b that was derived due to a flap opening of the closed form and which might help in accommodating RNA and other trans-acting factors within the U11/U12 di-snRNP (Chapter 4). In the current chapter, the SF3b open form and its interaction with the RNA elements is studied. The 5' end of U12 snRNA and its interaction with pre-mRNA in branch point duplex was modelled guided by the open form of SF3b that provided the necessary structural constraints and the RNA model is topologically consistent with the existing biochemical data. Further, utilizing the SF3b opens form-RNA model and the existing experimental knowledge, an extensive discussion has been provided on how the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation as well as its potential implications for branch point adenosine recognition fidelity. Moreover, the reasons for SF3b to be defined as a “fuzzy” complex - a complex with highly flexible folded regions along with intrinsically disordered regions is also discussed. Hence, the current work adds to the excellent developments made previously and deepens the understanding of the structure-function relationship of the human SF3b complex in the context of the spliceosome machinery. In chapter 6, a methodology has been proposed for the use of evolutionary conservation of protein-protein interfacial residues in multiple protein cryo-EM density based fitting of the protein components in the low-resolution density maps of multi-protein assemblies. First, the methodology was tested on a dataset of simulated density maps generated at four different resolutions -10, 15, 20 and 25 Å. On utilizing the evolutionary conservation scores obtained from multiple sequence alignments to score the fitted complexes, it was found that there was a decrease in the conservation scores when compared to that of the crystal structures, which were used to generate the simulated density maps. Further, the assessment of the multiple protein density fitting technique to align the actual protein-protein interface residues correctly using a performance metric called F-measure showed there was a decrease in performance as the resolutions became poorer. Hence, based on evolutionary conservations scores as well as F-measure the decrease in conservation scores or performance was found to be mainly due to the errors associated with the fitting process. Subsequently, a refinement methodology was designed involving the use of conservation scores, which improved the accuracy of the fitted models and the same, was observed in an experimental cryo-EM density test case of RyR1-FKBP12 complex. Hence, the conservation information acts as an effective filter to distinguish the incorrectly fitted structures and improves the accuracy of the fitting of the protein structures in the density maps. Thus, one can incorporate the conserved surface residues information in the current density fitting tools to reduce ambiguity and improve the accuracy of the macromolecular assembly structures determined using cryo-EM. In the concluding chapter 7, the learnings on the structural and mechanistic features of protein assemblies obtained from the use of computational techniques and integration of experimental datasets is discussed. In chapter 2, the modelling of a binary macromolecular complex such as the Gcn1-Gcn2 complex was performed using computational structure prediction strategies to understand the molecular basis of its interaction. Due to the potential inaccuracies which can exist in computational modelling, the chapters 3 to 5 dealt with the use of integrative approaches, primarily guided by the cryo-EM map, in order to decipher the molecular architecture of the human SF3b complex in the closed and open forms as well as its contribution for branch point adenosine recognition. Based on the extensive experience gained in modelling of assemblies using cryo-EM data in the previous chapters, a new method has been proposed on the use of evolutionary conservation information to improve the accuracy of cryo-EM density based fitting. Hence, these studies have provided strategies for modelling macromolecular assemblies as well as a deeper understanding of its mechanistic features.

Le travail présenté dans ce manuscrit a montré l’importance du développement méthodologique et technique pour identifier et débloquer les verrous empêchant l’analyse de matériaux hybrides et complexes qui se dégradent sous irradiation par un faisceau d’électrons. Nous avons mis en évidence que des dégâts sur l’échantillon produits par les électrons n’apparaissent qu’au-dessus d'un certain seuil de densité de courant électronique qui dépend de la nature du matériau et de ses caractéristiques morphologiques et structurales. Ces développements couplés à la Cryo-EM, nous ont permis de mettre en évidence l’architecture des matériaux hybrides à base de carbone, la variation de la distance lamellaire dans une pérovskite en fonction de la molécule insérée et le positionnement du métal, d’identifier les interactions à l’interface entre deux cristaux moléculaires et la quantification 3D de la fonctionnalisation d’un MOF. Dans la dernière partie, nous avons mis en évidence les processus de nucléation et de croissance d’oxyde de fer par MET in-situ en phase liquide. / The present manuscript shows the importance of methodological and technical development to identify and to unblock locks preventing the analysis of hybrid and complex materials that undergo degradation under electron beam irradiation. We have shown that beam-induced damage to the sample only appears above some specific threshold of current density. Such a threshold depends on the nature of the material and on its morphological and structural characteristics. These developments in synergy with the use of Cryo-EM, allowed us to expose the architecture of carbon-based hybrid materials, measure the variation of the lamellar distance in a perovskite according to the molecular spacer and to the positioning of the metal, identify the interactions at the interface between two molecular crystals, and the 3D quantification of the functionalization within a MOF. Lastly, we brought to light the processes of nucleation and growth of iron oxide by in-situ liquid phase TEM.

Cell cycle regulation is an important part for the detection and destruction of mutated and dysregulated cells as it is a natural protection against degenerative diseases and cancer. The ability of the body to detect and destroy these cells is a vital part in maintaining homeostasis in the body. Once cells have circumvented this line of defence, dismantling these cells would become very difficult.  Research into new ways to target and destroy mutated cells are constantly evolving in hopes of being able to control and direct lysis of target cells using therapeutic drugs. One of the possibilities for such a method are Cholesterol Dependent Cytolysins (CDC), specific proteins found in bacteria. These proteins are dependent on the ability to bind to cholesterol in cell-walls to form pores that lyse and effectively destroy cells.   This project aims to study the structure and mechanistic details of pore formation by CDC mitilysin using cryogenic electron microscopy (cryo-EM). Mitilysin was purified by affinity chromatography and its pore formation ability was confirmed by calcein release assay and hemolysis assay. The pore structures of mitilysin were observed by transmission electron microscopy (TEM) using liposomes composed of both 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as model membranes. Detergent screening directed separation of pores from liposomes; so that they could be visualized by cryo-EM. While these steps were optimized and proven successful, they were time-consuming. An initial 3D-model of pore-structures was rendered, but no molecular characteristics could be determined at the end of the allotted time. The study does lay the ground steps for obtaining the complete structure of mitilysin pores.

Translation is the process by which proteins are synthesized from the instructions in the genetic code. Translation is mediated by the ribosome, a large ribonucleoprotein complex, in concert with messenger RNA (mRNA), transfer RNA (tRNA), and a variety of proteins. The canonical mechanism of translation, introduced in Part I of my thesis, is divided into four distinct phases: initiation, elongation, termination, and recycling. Under unusual circumstances, each phase of translation can also proceed via a number of noncanonical mechanisms, many of which are vitally important for cellular growth or viral infectivity. My thesis describes structural insights into two such noncanonical mechanisms. The aim of the first project, described in Part II, was to structurally characterize a noncanonical mechanism of translational termination in bacteria. In the absence of a stop codon, ribosomes arrest at the 3′ end of an mRNA and are unable to terminate. In bacteria, the primary mechanism for rescuing such nonstop complexes is known as trans-translation. In the absence of a functional trans-translation system, however, the small protein ArfA recognizes the empty mRNA channel and recruits the release factor RF2 to the ribosome, enabling termination to occur. Using single-particle electron cryomicroscopy (cryo-EM), I obtained four high-resolution structures of nonstop complexes that reveal the mechanism of ArfA-mediated ribosome rescue and have wider implications for understanding canonical termination in bacteria. The aim of the second project, described in Part III, was to gain structural insights into a noncanonical mechanism of translational initiation in eukaryotes known as internal ribosome entry. Instead of a 5′ cap, many viruses contain intricately structured, cis-acting internal-ribosome-entry sites (IRESs) within their genomes that direct end-independent initiation. The IRES of hepatitis-C virus (HCV), for example, interacts directly with the mammalian ribosome and functionally replaces many of the canonical initiation factors. However, the mechanism by which the HCV IRES coordinates assembly of an initiation complex and progresses through the initiation phase remains poorly understood. I developed a method for purifying native ribosomal complexes from cell lysate that enabled me to obtain multiple cryo-EM maps of the HCV IRES in complex with the 80S ribosome, including a previously unseen conformation of the IRES induced by rotation of the ribosomal small subunit, and to make progress towards capturing earlier steps in the initiation pathway.

No description available.

Le ribosome est une machinerie cellulaire importante impliquée dans la synthèse protéique de toute cellule vivante. Par conséquent, le ribosome est l'une des principales cibles des antibiotiques naturels, qui sont capables de tuer les cellules bactériennes en bloquant la synthèse protéique. Toutefois, certaines bactéries sont résistantes à ces antibiotiques en raison de petites modifications au niveau de leurs ribosomes. Entre autres, Staphylococcus aureus (S. aureus) est un agent pathogène responsable de nombreuses infections graves chez l’Homme. Les structures cristallines d'antibiotiques en complexe avec des ribosomes de bactéries non-résistantes, non-pathogènes, Gram négatives ont fourni un aperçu sans précédent des mécanismes d'action de ces antibiotiques. Cependant, aucune structure de ribosome de bactéries pathogènes, hautement résistantes, Gram positives telles que S. aureus n’a encore été identifiée.Dans cette étude, nous présentons la première structure de ribosome de S. aureus à haute résolution (3.9 Å) résolue par cryo-microscopie électronique (cryo-ME). Nous mettons en évidence plusieurs caractéristiques de l'organisation des ribosomes spécifiques des bactéries Gram-positives. Nous décrivons également le protocole de purification et de cristallisation du ribosome de S. aureus pour de futures études de cryo-ME et de cristallographie aux rayons X.Tous les résultats obtenus dans ces travaux, faciliteront la description à l’échelle atomique du ribosome de S. aureus et ses complexes fonctionnels’ ’dans un futur proche. La combinaison des méthodes de cristallographie aux rayons X et de cryo-ME aidera à atteindre cet objectif. Les résultats obtenus serviront de base pour le développement de nouveaux composés contre la bactérie pathogène et extrêmement résistante qu’est S. aureus. / The ribosome is a large cellular machinery that performs the protein synthesis in every living cell. Therefore, the ribosome is one of the major targets of naturally produced antibiotics, which can kill bacterial cells by blocking protein synthesis. However, some bacteria are resistant to these antibiotics due to small modifications of their ribosomes. Among them, Staphylococcus aureus (S. aureus) is a severe pathogen that causes numerous infections in humans. The crystal structures of complexes of antibiotics with ribosomes from Gram-negative non-pathogenic non-resistant bacteria have provided unparalleled insight into mechanisms of antibiotics action. However, the structure of the ribosome from Gram-positive pathogenic and highly resistant bacteria such as S. aureus was still unidentified.In this study we present the first high resolution structure of the ribosome from S. aureus solved at 3.9 Å by cryo-electron microscopy (cryo-EM). We demonstrate several features of the ribosome organization which are unique for Gram-positive bacteria. We also describe the protocol of purification and crystallization of S. aureus ribosome for future cryo-EM and X-ray crystallography studies.All the results obtained in this work will help to describe S. aureus ribosome and its functional complexes at the atomic level in the nearest future. The combination of X-ray crystallography and cryo-EM methods will help to achieve this aim. The obtained results will provide a foundation for the development of new compounds against the pathogenic and extremely resistant bacteria S. aureus.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23113号 / 医科博第124号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 篠原 隆司, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Currently, human norovirus is the leading cause of acute gastroenteritis and accounts for most cases of foodborne illnesses in the United States each year. Due to its tissue culture inefficiency, studies of human norovirus have been crippled for more than forty years.Tulane virus (TV) stands out as a suitable surrogate of human norovirus given its high amino acid identity with human norovirus and its well-established cell culture system. It was first isolated from rhesus macaques (Macaca mulatta) in 2008 and identified as a novel Calicivirusrepresenting a new genus, Recovirus genus (Farkas et al., 2008). However, there are still unanswered questions about its infectious cycle and the essential factors for its infection. In this study, we have obtained a TV variant (the 9-6-17 strain) that has lost the binding ability to the B-type histo-blood group antigen (HBGA), which was proposed to be the receptor of both TV and human norovirus. In the first chapter, we outline how the sequence analysis,structural biology studies, and mutagenesis studies of the 9-6-17 TV strain have shed light on the interaction with its host cell receptor. To investigate the key residues for HBGA binding, we established the full-length infectious clone of the 9-6-17 TV strain. We present a highly selective transformation of serine 367, located in the predicted HBGA binding site, into a lysine residu e. Our results advance the understanding of genetic changes in TV required for adaptation to cell culture environments. Cryo-EM is an awarding winning technique that has been the greatest scientific breakthrough in recent years. It was awarded the Nobel Prize in Chemistry in 2017. Despite the technological advances of the direct electron detector and image processing software, several major roadblocks remain for high-resolution structure determination with cryo-EM. In the later chapters, we explored the most efficient way of using VPP to enhance image contrast, how to tackle the airwater interface problem by encapsulating target protein, how to reach a higher resolution by refining high order parameters, and the helical indexing problem in real space. These technical advances would benefit the whole cryo-EM community by providing convenient tools or insights for future directions.