Academic literature on the topic 'Cryo-EM structures'

In eukaryotic translation initiation, eIF2B, a 295 kDa multisubunit (from α to ε) complex,is the guanine nucleotide exchange factor (GEF) of eIF2, a GTP binding protein, and hasmultiple roles in regulating the level of active eIF2-GTP-Met-tRNAi ternary complexes inthe cytoplasm. Mutations in eIF2B subunits affect global protein synthesis and, in human,are responsible to cause a genetically inherited lethal childhood brain disease calledLeukoencephalopathy with Vanishing White Matter (VWM). Although the genetic aspectseIF2B have been widely studied over decades, detailed structural knowledge only becameavailable in recent years but is still limited. This study aims to gain structural insights intoyeast eIF2B by a range of electron microscopy techniques to improve our understandingtowards its GEF activity with eIF2 and regulatory response. By performing size-exclusion chromatography and multi-angle static light scattering (SECMALS), it was found that eIF2B is a stable dimer of pentamers (~600 kDa). Negativestaining (25.8 Å) and cryo-EM (12.1 Å) eIF2B decamer models that showed 2-foldrotational symmetry were generated by single particle reconstruction. Homology modelingof yeast eIF2B subunits revealed an eIF2B(αβδ)2 hexameric core and two separate arm-likeeIF2Bγε catalytic domains with potential flexibility. To constrain subunit position in thearm structure, Ni-NTA-Nanogold labeling against the multihistidine tag of eIF2Bγ wasperformed. In addition, genetic approaches were applied to eliminate synthesis of eIF2Bα(34 kDa) and eIF2B(βγδε)2 octamer complexes (532 kDa) were purified by SEC-MALSand analysed by negative staining single particle reconstruction. It was speculated thatdeletion of eIF2Bα might have triggered significant conformational rearrangement that ledto high uniformity in the 2D class averages. A hypothetical model was thus proposed forthe octamer where the two arm-like domains clamp together to form a compact structure.

Ma thèse se focalise sur la compréhension d’un phénomène de transcription, appelé backtracking, qui inactive la RNAP et arrête la transcription. La réactivation des complexes RNAP arrêtés et la reprise de la transcription nécessitent un facteur protéique appelé GreB. L’objectif du projet était d’obtenir des informations structurelles sur: i) la façon dont le retour en arrière inactive la RNAP dans E. coli; et ii) comment GreB sauve la RNAP en marche arrière pour continuer la transcription. À l'aide de SP cryo-EM, j’ai capturé quatre instantanés de RNAP dans différents états. Mes résultats montrent que l'ARN n'est plus aligné avec le site actif. De plus, suite à un retour en arrière, la RNAP adopte de nouvelles modifications de conformation permettant la liaison de GreB. En conséquence, le NTD de GreB entre en contact le site actif de la RNAP et donne des résidus acides qui augmentent l'affinité pour un ion magnésium, ce qui est nécessaire pour la catalyse du clivage de l'ARN mal aligné. Ces quatre reconstructions donnent un aperçu du mécanisme catalytique et de la dynamique du clivage et de l'extension de l'ARN<br>[...]My Ph.D. was focused on the understanding of a transcriptional phenomenon, termed backtracking, which inactivates RNAP and halts transcription. Reactivation of halted RNAP complexes and transcription resumption, requires a protein factor called GreB. The objective of the project was to gain structural information on: i) how backtracking inactivates RNAP inE. coli; and ii) how GreB rescues backtracked RNAP to continue transcription. Using SP cryo- EM, I captured four snapshots of RNAP at different states covering the backtracking and reactivation cycle. My results show that the RNA is no longer aligned with the active center, explaining the transcription halt. Furthermore, as a result of backtracking, RNAP adopts new conformational changes allowing GreB binding. As a consequence, the NTD of GreB contacts RNAP active center and donates acidic residues that increase the affinity towards a magnesium ion, which is required for cleavage catalysis of the misaligned RNA. These four reconstructions give insights on the catalytic mechanism and dynamics of RNA cleavage and extension. [...]

Les kinétoplastides sont un groupe de protozoaires, et qui menace plus de 400 millions de personnes dans le monde entier. Ils possèdent des segments d'expansion d'ARNr (SE) inhabituellement plus larges dans les sous-unités 40S. Ici, nous avons purifié à partir de lysats de cellules de T. cruzi des complexes d'initiation natifs (48S IC) et des sous-unités de 40S natives que nous avons ensuite analysées par cryo-ME. La structure des 48S IC révèle certains des aspects spécifiques de la traduction aux kinétoplastides, tels qu'un réseau d’interaction complexe entre eIF3 et SEs. En outre, notre structure met en évidence le rôle de DDX60 dans l'initiation de la traduction chez les kinétoplastides. La structure d'une sous-unité 40S native révèle l'existence d'un facteur non caractérisé (appelé ηF). Le site de liaison de ηF suggère un rôle dans le contrôle de la traduction. De plus, nous avons rapporté́ la structure d’une nouvelle protéine ribosomale (-r) spécifique des kinétoplastides (KSRP). Notre travail pose les premières bases structurales des aspects spécifiques de l'initiation de la traduction chez les kinétoplastides<br>Kinetoplastid is a group of flagellated protozoans, which threatens more than 400 million people world-wide. They possess unusual large rRNA expansion segments (ES) in the 40S, such as ES6S, ES7S and ES9S and their location suggests an involvement in the initiation process. Furthermore, all mature mRNAs possess a conserved 5’ spliced-leader. Here, we purified from T. cruzi cell lysates native initiation complexes and native 40S subunits that we then analysed by cryo-EM. The structure of native initiation complexes reveals several kinetoplastid-specific aspects of translation, such as an intricate interaction network between eIF3 and ES6S and ES7S. Furthermore, it reveals the role of DDX60 in translation initiation in kinetoplastids. The structure of native 40S subunits reveals the existence of an uncharacterized factor (termed ηF) bound at platform of the 40S. The binding site of ηF suggests a role in translational control. Moreover, we reported a novel kinetoplastid-specific ribosomal (r-) protein (KSRP) bound to the 40S subunit. Our work represents the first structural characterization of kinetoplastids-specific aspects of translation initiation

The mitochondrial F-ATPases make about 90% of cellular ATP. They are multi-protein assemblies with a membrane extrinsic catalytic domain attached to a membrane embedded sector. They operate by a mechanical rotary mechanism powered by an electro-chemical gradient, generated across the inner mitochondrial membrane by respiration. A detailed molecular description has been provided by X-ray crystallographic studies and "single molecule" observations of the mechanism of the F1 catalytic domain. Details are known also of the architecture of the peripheral stalk of part of the stator and the membrane embedded region of the rotor. However, knowledge of the detailed structure of the rest of the membrane domain, and the detailed mechanism of generation of rotation is lacking. Recently, studies of the intact mitochondrial F-ATPases, determined by cryo-electron microscopy (cryo-em), have provided structural information at intermediate levels of resolution. Whilst these structures have given insights into the mechanism of generation of rotation, the information required for a molecular understanding of this mechanism is still lacking. Moreover, the locations and roles of six supernumerary membrane subunits are unclear. Some of them are likely to be involved in the formation of dimers of the enzyme which line the edges of mitochondrial cristae. Therefore, in this thesis, a procedure is described for the purification of dimers of the bovine and yeast F-ATPases. The structure of the bovine dimer has been determined by cryo-em at a resolution of ca. 6.9 Angstrom. This structure confirms features concerning the trans-membrane spans of the a-, A6L- and b-subunits observed in the monomeric complex. In addition, the single trans-membrane a-helix of the f-subunit has been located, and the subunit appears to mediate dimer formation. The structure of A6L has been extended, and the a-helices of subunits e- and g- have been located. Another novel feature has been assigned to the DAPIT subunit, and may provide links between dimers in forming larger oligomers. Further improvement in the resolution of the structure is hampered by the extreme conformational heterogeneity of the F-ATPase. To this end, the simpler Fo membrane domain has been isolated and characterized initially by electron microscopy in negative stain.

Helical assemblies of proteins are ubiquitous in nature and they perform vital functions in a wide range of organisms. The recent development of direct electron detectors and other imaging techniques in cryo-electron microscopy (cryo-EM) has opened new possibilities in solving helical structures at atomic resolution. Existing software packages for helical processing often require experience in tuning many ad hoc parameters to achieve optimal reconstruction results. REgularised LIkelihood OptimisatioN (RELION), an open-source single-particle analysis package, reduces the need for user expertise by the formulation of an empirical Bayesian framework, and has yielded some of the highest resolution density maps in recent years. Prior information about the helical assemblies can be conveniently incorporated into the statistical framework of RELION and thereby improves the helical reconstructions. This PhD thesis describes the development of a helical processing computation workflow with reduced user intervention in RELION. Chapter 1 introduces the theoretical basis of cryo-EM data acquisition and single-particle data processing, the concepts of helical symmetry, and a previously described method for iterative real-space reconstruction of helical assemblies, to which the RELION implementation bears resemblance. Chapter 2 discusses multiple adaptations to RELION that are necessary for helical processing. Key elements include the imposition and local refinement of helical symmetry, masks on helical segments and references, expressions of angular and translational prior information, manual and automated segment picking as well as initial model generation for helices. Calculations have been performed on four test data sets showing that the developed methods in RELION yield results that are as good as or better than alternative approaches for the tests performed. Chapter 3 describes the same methodology adapted to helical sub-tomogram averaging in RELION. Chapter 4 introduces the local symmetry option developed for special types of filaments with pseudo-helical symmetry. The concept can be extended to general single-particle analysis as well. Chapter 5 describes four helical structures determined in collaboration with other research groups using helical RELION for data processing. Chapter 6 concludes the thesis with a brief summary and future prospects.

Une approche de biologie structurale intégrative a été mise à profit pour l'étude de l’organisation structurale du complexe NuRD. Mon travail s'est focalisé essentiellement sur trois sous-unités du complexe: MBD3, RbAp46 et RbAp48. J'ai mis en place les protocoles de production et de purification de ces différentes sous-unités, et les ai caractérisé biophysiquement par diverses méthodes. Nous avons ensuite entrepris des études de liaisons sur des nucléosomes reconstitués au laboratoire. Pour MBD3, l'optimisation du complexe nous a permis d'obtenir des cristaux diffractant jusqu'à 7 A de résolution. Parallèlement, une reconstruction 3D préliminaire à partir de données de cryo-microscopie électronique a pu être obtenue à 25A de résolution. Pour RbAp46/48, nous avons pu montrer que ces protéines formaient un complexe stable avec le nucléosome, pavant la voie pour leur future étude structurale par cryo-microscopie électronique ou cristallographie aux rayons-X<br>An integrative structural biology approach has been used to study the structural organization of the NuRD complex.My work focused especially on three subunits of this complex: MBD3, RbAp46 and RbAp48. I set up the preparation of the individual subunits and characterized them by various biophysical methods. We next carried out binding assays with homemade human nucleosomes. For MBD3, optimization of the complex led to crystals diffracting up to 7 Å. In parallel, a preliminary 3-D reconstruction at 25 Å resolution has been solved in cryo-EM. For RbAp46/48, crystal we were able to show that these proteins form stable complexes with the nucleosome, paving the way for future structural analysis by cryo-EM or X-ray crystallography

La pause transcriptionnelle marquée par les ARN polymérases (RNAP) est un mécanisme clé pour réguler l'expression des gènes dans tous les règnes de la vie et est une condition préalable à la terminaison de la transcription. Le facteur de transcription bactérien essentiel NusA stimule à la fois la pause et la terminaison de la transcription, jouant ainsi un rôle central. Ici, je présente des reconstructions par cryo-microscopie électronique (cryo-EM) à une seule particule de NusA lié à des complexes d'élongation en présence et en absence d’ARN en épingle à cheveux dans le canal de sortie de l'ARN. Les structures révèlent quatre interactions entre NusA et RNAP qui suggèrent comment NusA stimule le repliement de l’ARN, la pause et la terminaison de la transcription. Un intermédiaire de translocation asymétrique de l'ARN et de l'ADN convertit le site actif de l'enzyme en un état inactif, fournissant une explication structurelle pour l'inhibition de la catalyse. La comparaison de RNAP à différentes étapes de la mise en pause donne un aperçu de la nature dynamique du processus et du rôle de NusA en tant que facteur de régulation<br>Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, I present single-particle electron cryo-microscopy (cryo-EM) reconstructions of NusA bound to paused elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor