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Etude structurale de macromolécules biologiques par cryomicroscopie électronique, reconstruction tridimensionnelle et recalage de données de cristallographie aux rayons X

Abstract : In biostructure, the discipline of cryoelectron microscopy allows to obtain high resolutions, with specific methods and techniques. In this context, my work focused on the study of one category of molecules, isolated particles with low degree of symmetry, and particularly on respiratory extracellular pigments such as cephalopod hemocyanins (Vampyroteuthis infernalis, Benthoctopus species, Sepia officinalis) and an annelid hemoglobin (Lumbricus terrestris). An empiric approach was selected, and permitted us to differentiate technical information from biological results. The first goal of my thesis was to use a known biological material to work on the different steps of cryoelectron microscopy : samples observation, images analysis, reconstruction, and correction of 3D structures. The second goal was to bring biological answers through these technical advances. At the beginning of my thesis, I used a microscope equipped with a LaB6 crystal, limiting the resolution at a value of 20 Å. Nevertheless, it allowed us to answer to a phylogenetic question (Benthoctopus species and Vampyroteuthis infernalis hemocyanins belong to sister orders), and to demonstrate the role of first, the type of microscope, and second, the electron source. Naturally, the next step was to access to a microscope equipped with a field emission gun. The short periods of access to this type of microscope, the first one is arrived in France in 2000, did not give us the possibility to systematically test all the modes. In spite of this problem, we obtained results that confirmed the advantages of the field emission gun. The boundary of 6-7 Å resolution, corresponding to the partial access to information describing the secondary structure of proteins, is not yet reached, but it is clear for this type of particles that we are on the good way. This progression is partially based on the evolution of methods of images collecting, reconstruction and correction of contrast transfer function.
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Submitted on : Wednesday, June 9, 2004 - 11:25:32 AM
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  • HAL Id : tel-00006224, version 1

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Fabrice Mouche. Etude structurale de macromolécules biologiques par cryomicroscopie électronique, reconstruction tridimensionnelle et recalage de données de cristallographie aux rayons X. Biochimie [q-bio.BM]. Université Pierre et Marie Curie - Paris VI, 2001. Français. ⟨tel-00006224⟩

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