This Program Project represents a coordinated effort to obtain high resolution structures of protein crystals by the method of electron crystallography. Electron crystallography is distinct from x-ray crystallography in that the use of electrons requires very thin, two- dimensional crystals, preferably protein monolayers, while crystals that are typically 100 mum thick, or more, are required for x-rays. The majority of the work proposed here will focus on membrane proteins, which have proven to be rather difficult to crystallize for x-ray diffraction. Membrane proteins can be expected a priori to form two-dimensional crystals when confined to the plane of a phospholipid bilayer, however, and therefore they represent a natural group of proteins for which electron crystallography can play a role of great biological importance. Project A will work with bacteriorhodopsin [an important ion (H+) pump and an analog of the superfamily of 7-transmembrane helix receptor proteins] to advance the ability of electron crystallography to obtain three- dimensional structures from the current level of about 3.5 Angstroms resolution to the goal of about 2.5 Angstroms resolution. Project B will use crystallographic difference Fourier methods to probe the conformational changes that occur in bacteriorhodopsin as part of its H+- pumping photocycle, in order to better understand the biochemical mechanism involved in this particular example of electrogenic ion transport. Project C will exploit recent progress that has been made towards obtaining well-ordered, two-dimensional crystals of erythrocyte membrane Band 3 (anion exchange) protein, to initiate a crystallographic structure analysis of this protein. Project D represents the effort of an Associate-member PI (work pursued with existing funding) to obtain crystals of two bacterial membrane proteins which would be suitable for electron crystallographic structure analysis. Project E will exploit the recent cloning, expression and purification of the important beta2- adrenergic membrane receptor. The CORE of the Program Project provides support for large, expensive equipment needed in electron crystallographic structure analysis, as well as the supporting infrastructure for its maintenance and use. The CORE is also involved in providing further advances in existing technology. The key advance in instrumentation will be to provide direct electronic readout of spot-scan images and of electron diffraction patterns by means of a CCD camera whose design has been optimized for use at 400 keV. Advances in membrane protein expression will be explored by the investigation of new, immortalized cell lines that may make it possible to target the expressed protein to lung- surfactant; the goal is to avoid the problem of toxicity that has sometimes been identified with attempts to achieve high-level expression in other eukaryotic cell lines. Finally, a small, exploratory CORE research effort will attempt to broaden the number of opportunities to get two-dimensional crystals of soluble proteins by adsorption to lipid monolayers, thereby making the technique of high resolution electron crystallography a valuable addition to the field of structural biology in many other areas of cell and molecular biology beside cell membrane proteins.
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| Glaeser, Robert M (2018) PROTEINS, INTERFACES, AND CRYO-EM GRIDS. Curr Opin Colloid Interface Sci 34:1-8 |
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