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Aujourd'hui, la cristallographie de macromolécules produit couramment des modèles moléculaires à résolution atomique. Cependant, cette technique est particulièrement difficile à mettre en œuvre, dans le cas de complexes de taille importante. La microscopie électronique permet, elle, de visualiser des particules de grande taille, dans des conditions proches de celles in vivo. Cependant, la résolution des reconstructions tridimensionnelles obtenues exclut, en général, leur interprétation directe en termes de structures moléculaires, étape nécessaire à la compréhension des problèmes biologiques. Il est donc naturel d'essayer de combiner les informations fournies par ces deux techniques pour caractériser la structure des assemblages macromoléculaires. L'idée est de positionner les modèles moléculaires déterminés par cristallographie à l'intérieur de reconstructions 3D issues de la microscopie électronique, et de comparer la densité électronique associée à la reconstruction 3D avec une densité électronique calculée à partir des modèles. Le problème numérique réside dans la détermination et l'optimisation des variables qui spécifient les positions des modèles, considérés comme des corps rigides, à l'intérieur de l'assemblage. Cette idée simple a donné lieu au développement d'une méthode appelée recalage. Ce travail de thèse a eu pour but de fournir aux biologistes un outil, basé sur la méthode du recalage, qui leurs permette de construire des modèles pseudo-moléculaires associés aux assemblages produits par microscopie électronique. Le logiciel issu de ce travail, nommé VEDA est un environnement graphique convivial, intégrant la possibilité de recalage flexible, et un moteur de calcul performant (calcul rapide, traitement de symétries complexes, utilisation de grands volumes, ...). Testé sur des dizaines de cas réels, VEDA est aujourd'hui pleinement fonctionnel et est utilisé par un nombre croissant de chercheurs, en France et à l'étranger qui lui reconnaissent tous facilité d'utilisation, stabilité, rapidité et qualité des résultats. / Today, macromolecular crystallography commonly produces molecular models at atomic resolution. However, this technique is particularly difficult to implement in the cases of large complexes. Electron microscopy allows for the visualization of large particles under conditions similar to those in vivo. However, the resolution of three-dimensional reconstructions obtained by electron microscopy does not allow, in general, the direct interpretation of molecular structures, a necessary step in understanding biological problems. Therefore, it is natural to try to combine information provided by these two techniques to characterize the structure of macromolecular assemblies. The idea is to place the molecular models determined by crystallography inside electron microscopy 3D reconstructions, and compare the electron density associated to the 3D reconstruction with an electron density calculated from the models. The numerical problem is the determination and the optimization of the variables that specify the positions of the models, considered as rigid body, inside the assembly. This simple idea has led to the development of a method called fitting. This thesis aims to provide biologists with a tool, based on the method of fitting, that allows them to build pseudo-molecular models associated to the assemblies produced by electron microscopy. The software resulting from this work, named VEDA, has a user-friendly graphical environment, includes the possibility of flexible fitting, and implements a powerful calculation core (fast computation, treatment of complex symmetry, use of large volumes, etc.). Tested on dozens of real cases with experimental data sets, VEDA is now fully functional and is used by a growing number of researchers in France and abroad. All of the users have recognized ease of use, stability, speed, and quality of results of VEDA. STAR

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.