This project has its final goal to understand the structural changes in tubulin related to nucleotide hydrolysis and resulting in the dynamic nature of microtubules. To this aim, we will compare the structures of the tubulin dimer in two different conformation representing the high and low energy states of the protein: the conformation in a straight protofilament where the tubulin-GDP molecule is constrained in a """"""""GTP-like"""""""" state by the polymer lattice, and the conformation in rings where are curved protofilaments where tubulin-GDP is in a relaxed state. The structure of tubulin in the constrained state has been recently obtained in Downing's lab by electron crystallography of zinc-induced sheets. The present project will be concerned with obtaining the structure of the tubulin rings by cryo-electron microscopy of vitrified samples and image reconstruction using single particle methods. An immediate step will be to obtain a medium resolution (approximately 15 A) projection of the ring structure. The interpretation of the projection will be helped by comparison with the atomic model of the constrained tubulin in sheets. Some general differences between the two conformations of tubulin could already be gained at this stage, allowing us to test proposed hypothesis on the effect of nucleotide content on the structure of the tubulin dimer. The next steps will involves reconstructions of increasing resolution as the data collection and processing progresses. A reconstruction at 10 Angstroms will test us not only the overall change in the shape of the dimer, but whether there are any tertiary structure rearrangements in the molecule; which secondary structure elements are most affected by the nucleotide state; and how these structural changes may affect the assembly properties of tubulin.
| Downing, Kenneth H; Glaeser, Robert M (2018) Estimating the effect of finite depth of field in single-particle cryo-EM. Ultramicroscopy 184:94-99 |
| Nogales, Eva (2018) Cryo-EM. Curr Biol 28:R1127-R1128 |
| Sazzed, Salim; Song, Junha; Kovacs, Julio A et al. (2018) Tracing Actin Filament Bundles in Three-Dimensional Electron Tomography Density Maps of Hair Cell Stereocilia. Molecules 23: |
| Kamennaya, Nina A; Zemla, Marcin; Mahoney, Laura et al. (2018) High pCO2-induced exopolysaccharide-rich ballasted aggregates of planktonic cyanobacteria could explain Paleoproterozoic carbon burial. Nat Commun 9:2116 |
| Howes, Stuart C; Geyer, Elisabeth A; LaFrance, Benjamin et al. (2018) Structural and functional differences between porcine brain and budding yeast microtubules. Cell Cycle 17:278-287 |
| Glaeser, Robert M (2018) PROTEINS, INTERFACES, AND CRYO-EM GRIDS. Curr Opin Colloid Interface Sci 34:1-8 |
| Kellogg, Elizabeth H; Hejab, Nisreen M A; Poepsel, Simon et al. (2018) Near-atomic model of microtubule-tau interactions. Science 360:1242-1246 |
| Zhang, Rui; LaFrance, Benjamin; Nogales, Eva (2018) Separating the effects of nucleotide and EB binding on microtubule structure. Proc Natl Acad Sci U S A 115:E6191-E6200 |
| Nogales, Eva (2018) Cytoskeleton in high resolution. Nat Rev Mol Cell Biol 19:142 |
| Han, Bong-Gyoon; Watson, Zoe; Cate, Jamie H D et al. (2017) Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality. J Struct Biol 200:307-313 |
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