Electron crystallography has a key role to play in understanding integral membrane proteins (IMPs) as it provides one of the most direct means of providing insight into the functional state of these molecular machines in their lipid-associated forms, and also has the potential to facilitate examination of physiologically relevant transitional states and complexes. Helical or tubular crystals, which are the natural product of proteins crystallizing on the surface of a vesicle, offer some unique advantages over other crystalline forms. The goal of this proposal is to develop an integrated pipeline to enable structure determination by transmission electron microcopy of IMPs in the form of tubular crystals. Given the very high number of IMPs that need to be solved, helical crystallization should be available as a standard method in the armamentarium of approaches for determining the structure of IMPs. However, its use is currently limited by a lack of standardized approaches and the enormous effort and specialized expertise required that form a barrier to entry. New or streamlined approaches are needed in three areas: crystal growth, screening, and analysis. An integrated approach is essential to ensure that progress in any one area is not throttled by bottlenecks at significant other steps in the process. Our goal is to establish TEM of tubular crystals as a standard approach to complement X-ray crystallography and NMR for characterizing IMPs.
Membrane proteins account for ~30% of gene products in organisms and are the target of ~60% of all therapeutic drugs currently in development. This research will establish helical crystallization as a standard method to add to the armamentarium of approaches for determining the structure of these critically important macromolecular machines.
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