Core - Improved High-resolution cryo-EM Methodology Jamie Cate and Kenneth Downing PROJECT SUMMARY/ABSTRACT The capabilities of cryo-EM have begun to match or surpass those of X-ray crystallography in many areas of structural biology. However, there remain crucial bottlenecks in cryo-EM that hamper reconstructions of fragile complexes. The goal of this Core Project is to develop methods that will further enhance the capabilities of cryo-EM, in order to support the work of the Projects and Associated Projects of this Program. We address two key areas where single-particle cryo-EM requires further improvement. We will first address the cryo-preservation of delicate samples on cryo-EM grids. Many samples are subject to denaturation when exposed to the extensive air-water interface during blotting, or to a continuous carbon grid. To prepare specimens in a more structure-friendly way by avoiding specimen contact with the air-water interface, streptavidin monolayer crystals will be used as a specimen-support film for affinity-binding interactions. We will test a series of affinity adaptor and affinity tagging methods, to ensure uniform particle orientations on the streptavidin monolayer crystal lattice. These experiments will demonstrate the general utility of using streptavidin monolayer crystals as a support material. Second, we will develop means to correct beam-induced sample movement. When using direct electron detectors in ?movie? mode, the initial frames of exposures are presently discarded, although they potentially contain the highest resolution information. In subsequent frames, the high-resolution information is no longer available due to electron beam induced damage. We propose to use the image of the streptavidin support film as a fiducial, to better characterize the frame-to-frame motions that occur. We will use human 48S translation preinitiation complexes and Escherichia coli 70S ribosomes as test systems to validate the methods in these two aims. Once we have established these methods, they will be of wide use in the cryo-EM community, and should greatly expand the scope of cryo-EM into areas of structural biology that were previously inaccessible, or accessible only to x-ray crystallography.
| 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 |
| 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 |
| Jorgens, Danielle M; Inman, Jamie L; Wojcik, Michal et al. (2017) Deep nuclear invaginations are linked to cytoskeletal filaments - integrated bioimaging of epithelial cells in 3D culture. J Cell Sci 130:177-189 |
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