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The Center for High-Throughput Structural Biology (CHTSB) is one of six specialized research centers created in July 2005 as part of phase two of the National Institutes of Health’s Protein Structure Initiative (PSI). It is comprised of 11 laboratories at six institutions located in upstate New York, California, and Toronto, Ontario. The center’s mission is to overcome the most significant obstacles to structure determination by focusing on technology development in areas such as protein production, crystallization and cryo-preservation that are steps in sample preparation for X-ray diffraction studies. Newly evolved techniques are being applied in a medium-throughput structure determination pipeline to protein targets from baker’s yeast (Saccharomyces cerevisiae). These targets include transmembrane proteins (TMPs), protein:protein complexes, and scFv:TMP complexes that have traditionally been difficult to study by diffraction methods. It is anticipated that the technology developed within this initiative will aid in the establishment of a structural biology research resource that addresses the crystallization and structure determination needs of the greater biological community.