The structure of the dihydrolipoamide acetyltransferase (E2p) component of the pyruvate dehydrogenase complex from <i>Escherichia coli</i> and its role in catalysis were studied by the combined approaches of protein engineering, limited proteolysis and <SUP>1</SUP>H-n.m.r. spectroscopy. Genetic reconstruction of the E2p component (performed elsewhere) produced a series of mutant complexes assembled around E2p chains which contain only a single lipoyl domain and an associated (alanine+proline)-rich linker region of gradually diminishing lengths (32, 20, 13, 7 and 1 residue(s), respectively, in the pGS110-,pGS156-,pGS186 ,pGS187- and pGS188-encoded complexes). When this region was shortened to 13 residues or less, the system of active-site coupling in the enzyme complex was dramatically impaired, although the individual enzyme activities were unaffected. The role of the (alanine+proline)-rich region in facilitating moment of the lipoyl domains in catalysis was thus established. The (alanine+proline)-rich regions of the wild-type E2p chains had previously been conjectured to be the source of the unexpectedly sharp resonances in the <SUP>1</SUP>H-n.m.r. spectrum of the enzyme complex, and hence to be conformationally mobile. Examination of the genetically restructured complexes by <SUP>1</SUP>H-n.m.r. spectroscopy revealed that the intensity of the sharp peaks in the spectra correlated well with the length of the (alanine+proline)-rich region in each complex. Furthermore, resonances from a single histidine residue engineered into the (alanine+proline)-rich region of a pGS110-encoded E2p chain was clearly visible in the <SUP>1</SUP>H-n.m.r. spectrum of the resulting enzyme complex. These experiments proved unequivocally that the (alanine+proline)-rich regions are conformationally mobile. The <SUP>1</SUP>H-n.m.r. spectra of the mutant complexes with the most severe deletions in the E2p chains differed from those of the wild-type and pGS110-encoded complexes in that they displayed a novel sharp peak which was not initially apparent in the spectra of the parent assemblies. This resonance was tentatively assigned to another, shorter (alanine+proline)-rich sequence in the E2p chain, which separates the dihydrolipoamide dehydrogenase (E3)-binding and inner-core domains in the <i>C</i>-terminal half of the molecule. It is likely therefore that this sequence is also conformationally flexible. Antibodies against a synthetic peptide with the sequence of the long (alanine+proline)-rich region of the pGS110-encoded E2p chain were raised elsewhere. Binding of the Fab fragments of these antibodies to the pGS110-encoded complex was found to inhibit the overall complex activity even though the activities of the three component enzymes were not affected. Antibody binding was shown to prevent both the reductive acetylation of the lipoyl domains at the pyruvate decarboxylase (E1p) active site and the transfer of acetyl groups between adjacent lipoyl domains, demonstrating the role of the (alanine+proline)-rich sequence in the mechanism of substrate transfer between active sites. A detailed study of the conformation of the (alanine+proline)-rich regions was also undertaken. Synthetic peptides were obtained with sequences identical to the central and innermost such regions of the wild-type E2p chain. The conformation of these peptides in aqueous solution was studied by circular dichroism, <SUP>1</SUP>H-n.m.r. and <SUP>13</SUP>C-n.m.r. spectroscopy. Relaxation time and nOe data pointed to an extended conformation for the peptides, a structure enforced by the predominantly <i>trans</i> Ala-Pro peptide bond. The functional consequences of this conformation and the role of these sequences in the structure and the function of the enzyme complex are discussed.

Protein complexes mediate a diverse range of behavior in prokaryotic cells, yet the exact molecular mechanisms explaining how many of these complexes assemble and function remain unknown. This work focuses on understanding the molecular mechanisms of two different protein assemblies responsible for regulating virulence in the opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa utilizes type IV pili (T4P) to adhere to, and move along, surfaces. Assembly of T4P is powered by a dedicated cytoplasmic ATPase, PilB. The structural study of PilB from a related system (chapter 2) resulted in the formulation of the first model describing the mechanism of force generation resulting from ATP hydrolysis, which explains how T4P are assembled. Chapter 3 focuses on the RetS/GacS interaction, which is responsible for globally regulating virulence in P. aeruginosa. A comprehensive structural study reveals a dynamics of a novel regulatory interaction and the discovery of a potentially universal transmembrane signaling mechanism. / Doctor of Philosophy / Bacteria have threatened human health since the beginning of recorded history. With the development of antibiotics in the early twentieth century, the threat posed by bacterial infection was greatly lessened. However, decades of antibiotic mismanagement has led to the evolution of bacteria which are no longer vulnerable to these antibiotics. In order to combat this rising threat of resistant bacteria, we require a deeper understanding of how bacteria function and cause disease. Proteins play a crucial role in the diseases caused by bacteria, either by directly damaging host cells or regulating the expression of these damaging factors. By increasing our knowledge of the roles played by protein during bacterial infections, it will be possible to create new antibiotics while minimizing the risk of resistance. The work presented here grants a deeper understanding into how proteins work together to allow bacteria to survive inside the human body.

Les chaperonines sont des chaperonnes moléculaires indispensables pour le repliement de certaines protéines dans les cellules. La taille et la complexité de ces machineries biologiques rendent complexes l'étude de leurs propriétés structurales et fonctionnelles. La spectroscopie RMN permet de suivre des changements structuraux et dynamiques en temps réel avec une résolution atomique. Cependant, l'étude par RMN de protéines ou de complexes de haut poids moléculaires a été un challenge pendant de nombreuses années. Dans la première partie de cette thèse, il a été montré que la combinaison de marquage spécifique des groupements méthyles, d'expériences RMN optimisées et de microscopie électronique peut être utilisée pour suivre différents états du cycle fonctionnel d'une chaperonine de 1 MDa. Pour étudier ce mécanisme, la chaperonine native a été reconstituée avec un marquage des groupements méthyles des méthionines et valines. Les résidus méthionines ont pu être utilisés comme des sondes pour identifier les spectres RMN correspondant aux états intermédiaires et aux espèces actives du cycle fonctionnel. Grâce à ces sondes il a été possible de suivre en temps réel les réarrangements structuraux correspondant aux différentes conformations de la chaperonine durant son cycle fonctionnel. La seconde partie traite de la caractérisation de l'interaction de la chaperonine avec une protéine cliente dépliée. L'observation de la stabilisation de l'état déplié de la protéine par la chaperonine a permis de mettre en évidence une activité de "holdase" de la chaperonine. En utilisant une combinaison astucieuse de différents marquages de groupements méthyles et d'expériences RMN optimisés pour des assemblages de haut poids moléculaire, il a été possible d'observer le repliement de cette protéine par la chaperonine et les effets de la présence d'une protéine dépliée sur le cycle fonctionnel de la chaperonine en action. / Chaperonins are essential molecular chaperons for the refolding of proteins in the cells. Size and complexity of these biological machineries make complex the study of their structural and functional properties. NMR spectroscopy offers an unique ability to monitor structural and dynamic changes in real-time and at atomic resolution. However, the NMR studies of large proteins and complexes has been a real challenge for a long time. In the first part of this thesis, it has been shown that the combination of methyl specific labeling, optimized NMR spectroscopy for large assemblies and electron microscopy can be used to monitor the different states of the functional cycle of a 1 MDa chaperonin. To study this mechanism, the native chaperonin was reconstituted with a labeling of the methionines and valines methyl groups. Methionines residues have been used as probes to identify the NMR spectra corresponding to intermediates states and active species of the functional cycle. Thanks to theses probes, it has been possible to follow in real time the structural rearrangements corresponding to the different conformations of the chaperonin during its functional cycle. The second part deals with the characterization of the interaction between the chaperonin and an unfolded protein. Observation of the stabilization of the unfolded protein by the chaperonin allowed to identify the holdase activity of the chaperonin. Using a clever combination of a differential methyl labeling and optimized NMR spectroscopy for large assemblies, it has been possible to follow the refolding of the unfolded protein by the chaperonin and the effects of the unfolded protein on the functional cycle of the chaperonin in action.

L’étude de l’interaction entre des protéines et leurs ligands est essentielle pour la compréhension de leur rôle physiologique ainsi que de leur rôle dans la mise en place de pathologies. En particulier, dans le cas de maladies neurodégénératives, comme les maladies d’Alzheimer ou de Creutzfeld-Jacob, ou d’autres pathologies liées au mépliement de protéines, la compréhension des interactions entre protéines, qui peuvent modifier de façon considérable le paysage conformationnel des partenaires, est une des clés pour décrypter les étapes moléculaires élémentaires induisant une cascade d’événements qui mènent à la formation d’assemblages protéiques de grande taille. Dans le cadre de cette thèse, nous avons étudié la dynamique de taille de trois types d’assemblages protéines/ peptides.Interactions faibles impliquées dans la structuration d’assemblages de PrPSc et dans l’équilibre entre les formes PrPSc et suPrP : Dans un premier temps, l’étude des étapes précoces de la réplication du prion nous a permis de mettre en évidence une voie de diversification structurale qui implique une voie de templating secondaire. En utilisant une méthode de solubilisation en conditions natives et une approche reposant sur une série de souches différentes, nous avons démontré que, pour toutes les souches testées, les assemblages de prion ont la propriété de se dépolymériser en deux ensembles discrets d’assemblages oligomériques, dont la taille varie entre des dimères et tétramères. Des approches d’amplification de prion in vitro et in vivo ont démontré que ces assemblages oligomériques comportent toute l’information nécessaire à la réplication ainsi que les déterminants structuraux de la souche. Le motif de glycosylation, la cartographie par épitopes ainsi que l’empreinte PK ont mis en évidence des différences structurales entre les espèces oligomériques, soulignant la coexistence d’ensembles d’assemblages structuralement distincts au sein d’une souche donnée. Des expériences de dépliement partiel ont montré la présence d’une dynamique constitutionnelle entre les assemblages reposant sur un échange de matière et un réarrangement structurel à l’échelle de l’unité élémentaire (suPrP) ainsi qu’une diversité structurale au sein des assemblages de prion.Partenaires et oxydation de la PrPc: Bien que des travaux préalables aient mis en évidence l’existence d’interactions entre PrP et Aβ, Aβ1-40 ne forme que des complexes très instables avec la PrP monomérique. La spectrométrie de masse n’a pas permis de mettre en évidence cette interaction, même après optimisation des paramètres en conditions natives ou via des mesures indirectes reposant sur des échanges hydrogène - deutérium d’amide. L’oxydation radioloytique de la PrPC a été étudiée par SEC couplée à la mobilité ionique et la spectrométrie de masse, montrant la formation d’assemblages de haut poids moléculaires associée à des changements de structure.Fortes interactions peptide/peptide : Le système Synapt G2Si (Waters), qui combine de la mobilité ionique avec de la spectrométrie de masse, a été utilisé pour l’analyse d’une série de peptides issus de la capside externe du birnavirus de l’IBDV. Ces peptides peuvent former des complexes avec des énergies de liaison intermoléculaires proches de l’énergie de la liaison peptidique. En plus de la mise en évidence des changements structuraux au sein de ces complexes liés à l’introduction de mutations dans la séquence peptidique, des déviations par rapport aux attentes expérimentales ont été observées au cours de ce travail lors de mesures en conditions de mobilité ionique. Ces écarts ont pu être reliés à la conception de la cellule Tri-Wave de l’instrument, qui conduit à un mélange des différents gaz tampon qui ne peut être évité. De ce fait, les biais introduits par ce mélange influencent les paramètres de modélisation et la comparaison entre instruments pour tous les utilisateurs de ce modèle d’instrument. / Interactions between proteins and their ligands play a crucial in the understanding of their physiological function as well as their role in disease mechanisms. Especially in all forms of neurodegenerative diseases like Alzheimer, Creutzfeld-Jacob disease and others pathologies due to protein misfolding, the understanding of protein interaction, which can cause a dramatic change in the conformational landscape of the binding partners, is key in the deciphering of elementary molecular steps leading to cascade of events finally resulting in the formation of large protein assemblies. In the scope of this thesis we studied the size dynamic of three types of protein/peptides assemblies.Weak interactions involved in the structuration of PrPSc assemblies and in the detailed balanced between PrPSc and suPrP: The earliest steps in prion protein conversion were studied using both in vitro bona fide prion amplification method. We showed that the early step of the replication leads to a deterministic diversification process involving a secondary templating pathway. Furthermore, by using a native solubilization method and through a multi-strain approach. We demonstrated a generic property of prion assemblies to depolymerize into two discreet sets of oligomeric assemblies with a size ranging between dimers and tetramers for all tested strains. Both in vitro and in vivo prion amplification approaches showed that the oligomeric assemblies harbour all the replicative information as well as the strain structural determinant. Glycopattern, epitope-mapping and PK fingerprint revealed structural differences between the oligomeric species highlighting the coexistence of structurally distinct set of assemblies within a given strain. Partial unfolding experiments demonstrated the existence of a constitutional dynamic between the assemblies based on material exchange and structural rearrangement at the level of the elementary subunits (suPrP).PrPc partners and oxidative modifications: Although previous work had shown the existence of interactions between PrP and Aβ, monomeric Aβ1-40 forms only very weak complexes with monomeric PrP and mass spectrometry, even after optimization of the native instrument settings or by indirect measurements based on amide hydrogen deuterium exchange was not able to detect this interaction. Oxidation of PrPc was also suggested as a pathway towards pathology: the appearance and structural changes induced by gamma-radiolysis of water on PrPc were studied by mass spectrometry and ion mobility.Strong peptide/peptide interactions: The Waters Synapt G2Si instrumental set-up which couples ion mobility with mass spectrometry was used for the analysis of a collection of peptides derived from the shell of the IBDV binavirus. These peptides can form complexes with binding energies close or similar to the binding energy of the peptide bond. In addition to a better description of the changes in structure of these complexes varying with the mutations introduced in peptide sequence, we observed deviation from expected instrument characteristics when performing ion mobility measurements. Due to the design of the Tri-wave module in the Waters Synapt G2Si mass spectrometer, a mixture of the different buffer gases intrinsically cannot be avoided. Consequently, these biased measurements influence the outcome of modelling approaches and the inter-instrument comparison for all users of this instrument type.

No description available.

Ce travail de thèse porte sur le développement de méthodes en spectrométrie de masse (MS) et mobilité ionique (IM-MS) supramoléculaires pour la caractérisation fine de complexes protéine-ligand et d’assemblages protéiques hétérogènes de hauts poids moléculaires. L’optimisation instrumentale apportée à l’étude de ces systèmes, permet d’étendre le potentiel de ces deux approches en biologie structurale. Le criblage de complexes protéine-ligand permet ici une détermination de leurs propriétés d’interaction et la mise en évidence de subtils changements de conformation induits, pouvant être suivis au cours du temps. L’application de ce couplage à l’analyse de complexes multi-protéiques, réfractaires aux techniques conventionnelles, donne accès à la topologie de ces assemblages, facilitant la proposition de modèles structuraux. Enfin, l’apport récent de la haute résolution en MS native est ici illustré à travers l’étude de protéines complexes et hétérogènes : les anticorps thérapeutiques et leurs conjugués. Ces développements permettent de repousser certaines limites en MS native et IM-MS native, élargissant leurs perspectives d’application dans la recherche et l’industrie pharmaceutique. / This PhD thesis aims at developing methods in native mass spectrometry (MS) combined with ion mobility (IM-MS) to characterize protein-ligand complexes and large protein assemblies. Fine-tuning of instrumental settings allowed expanding the scope of these approaches in structural biology. Real-time monitoring of protein-ligand complexes by native MS and IM-MS enabled to screen their binding properties while depicting subtle conformational changes induced upon binding. Applying these methods to refractory multi-protein complexes provided insights about their topology, making structural modeling easier. Finally, benefits from high-resolution native MS were highlighted through the characterization of heterogeneous systems, including monoclonal antibodies and their drug conjugates. Here, these developments enable to push some technical limits one step forward, increasing the potential of native MS and IM-MS both in academic research and pharmaceutical industry.

La spectrométrie de masse, initialement développée pour de petites molécules, a permis au cours de la dernière écoulée d’étudier en phase gazeuse des assemblages macro-moléculaires intacts, posant nombre de questions algorithmiques difficiles, dont trois sont étudiées dans cette thèse. La première contribution concerne la détermination de stoichiométrie (SD), et vise à trouver le nombre de copies de chaque constituant dans un assemblage. On étudie le cas où la masse cible se trouve dans un intervalle dont les bornes rendent compte des incertitudes des mesures des masses. Nous présentons un algorithme de taille mémoire constante (DIOPHANTINE), et un algorithme de complexité sensible à la sortie (DP++), plus performants que l’état de l’art, pour des masses en nombre entier ou flottant. La seconde contribution traite de l’inférence de connectivité à partir d’une liste d’oligomères dont la composition en termes de sous-unités est connue. On introduit le problème d’inférence de connectivité minimale (MCI) et présente deux algorithmes pour le résoudre. On montre aussi un accord excellent entre les contacts trouvés et ceux détermines expérimentalement. La troisième contribution aborde le problème d’inférence de connectivité de poids minimal, lorsque chaque contact potentiel a un poids reflétant sa probabilité d’occurrence. On présente en particulier un algorithme de bootstrap permettant de trouver un ensemble d’arêtes de sensitivité et spécificité meilleures que celles obtenues pour les solutions du problème MCI. / Mass spectrometry (MS), an analytical technique initially invented to deal with small molecules, has emerged over the past decade as a key approach in structural biology. The recent advances have made it possible to transfer large macromolecular assemblies into the vacuum without their dissociation, raising challenging algorithmic problems. This thesis makes contributions to three such problems. The first contribution deals with stoichiometry determination (SD), namely the problem of determining the number of copies of each subunit of an assembly, from mass measurements. We deal with the interval SD problem, where the target mass belongs to an interval accounting for mass measurement uncertainties. We present a constant memory space algorithm (DIOPHANTINE), and an output sensitive dynamic programming based algorithm (DP++), outperforming state-of-the-art methods both for integer type and float type problems. The second contribution deals with the inference of pairwise contacts between subunits, using a list of sub-complexes whose composition is known. We introduce the Minimum Connectivity Inference problem (MCI) and present two algorithms solving it. We also show an excellent agreement between the contacts reported by these algorithms and those determined experimentally. The third contribution deals with Minimum Weight Connectivity Inference (MWCI), a problem where weights on candidate edges are available, reflecting their likelihood. We present in particular a bootstrap algorithm allowing one to report a set of edges with improved sensitivity and specificity with respect to those obtaining upon solving MCI.

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.