DESCRIPTION (provide by applicant): Helical crystals of proteins are ideal specimens for structure determination by electron microscopy. A new method for facilitating the crystallization of proteins as helical arrays on lipid-tubule substrates has been developed. This strategy has come at a unique time where high-resolution (near atomic) structures are becoming available by electron crystallography and the number of identified proteins for which no structural data exists has exploded. This also coincides with advances in computing power, image processing and correction algorithms, and automation of electron microscopy that will allow rapid structural analysis of helical crystals of proteins. The long-term objective of this work is to rapidly obtain structural information about proteins and macromolecular complexes to better understand the biological functions. To meet this challenge, the proposed research revolves around experiments to broaden the general utility of lipid-tubule substrates for helical crystallization of proteins. Specifically, novel lipids that can serve to bind recombinant proteins through specific molecular interactions will be produced and tested. These novel lipids will be thoroughly characterized for their capacity to be incorporated into lipid-tubules, specifically bind proteins, and facilitate the formation of helical arrays of proteins on lipid-tubules. Another important component of the proposed research involves studies to define the practical limits and capacities of this new technique. To test the resolution limits of this approach, a three-dimensional (3-D) structure of a relatively small protein (-60 kD) will be determined that can be compared to an established atomic model of the same molecule. Also, the capacity of this method to reveal information about conformational changes in a large protein complex will be tested by obtaining 3-D structures of the complex in different nucleotide-associated states. It is envisioned that this work will ultimately expand the range of protein structures that can be determined.
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