Small angle x-ray scattering studies of biological macromolecules at cryogenic temperatures

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A protein's biological job is related to its shape, thus insight can be gained into a protein's function by measuring its structure and how this structure changes as the protein interacts with other molecules. This task is challenging because many proteins are small, with diameters about ten thousand times less than the width of a human hair. The field of structural biology relies on exploiting experimental methods that provide accurate structural information on such small length scales. In the last decade, thanks to the availability of high intensity x-ray beams at national facilities (synchrotron sources) as well as the development of sophisticated analysis programs that return structural envelopes, small angle x-ray scattering (SAXS) has become a reliable tool for rapidly obtaining structures of proteins and other large biological molecules. The greatest strength of SAXS is its ability to report structures of biological macromolecules insolution, without the need for specialized sample preparation. A protein's shape can now be determined in a simple and straightforward way. However, the radiation used to measure the sample can also damage it, changing its structure. This problem has been encountered in other radiation-based methods, including x-ray crystallography and electron microscopy. For these latter fields, the use of cryo-freezing has reduced damage to tolerable levels: biological samples are protected from radiation at temperatures of ~100 K. The goal of this project is to design and construct two instruments that will prepare flash-frozen samples optimized for SAXS experiments: cryo-SAXS. Because frozen samples can tolerate longer exposure to radiation, without concern for radiation-induced structural changes, this method will allow structures to be obtained from even small amounts of precious sample. The successful demonstration of cryo-SAXS will garner tremendous interest within the large structural biology community. All plans/designs generated as a result of this project will be made public, through journal publication, presentations at conferences, posting on existing public forums for crystallography and SAXS, and webinars. Dissemination through use at synchrotron sources is also extremely effective, as a large number of users pass through and are exposed to new technologies through users' meetings, facility websites and workshops. A long-term goal of this project is to develop a kit that makes this technology easily available to the structural biology community.