Macromolecular crystallography remains the most important tool for experimental determination of three-dimensional structures of large biomolecules. From knowledge of structures, researchers can derive functional information, deepen understanding of macromolecular interactions, develop tools for protein engineering, and design ethical pharmaceuticals. The central hypothesis underlying this work is that the structure of a macromolecule in its crystalline form resembles its structure in a biological setting, and that the linkage between that structure and the macromolecule’s function is deep and informative. The specific systems Prof. Howard will study are mammalian citrate synthase, the first enzyme in the tricarboxylic acid cycle; and three understudied human proteins of clinical significance, namely GNB3, OTUD6B, and DDAH2. Prof. Howard hosted a student in the 2023 IIT X-ray REU program, Wendy Alvarez. She participated in crystallization of novel forms of citrate synthase and GNB3. He expects to obtain diffraction data from those crystals during academic year 2023-24. He continues to re-determine high-resolution structures already deposited in the Protein Data Bank with an eye toward deepening understanding about the role of solvent molecules in those structures. 

The specific research goals for one or two students participating in this project are:

·         To grow crystals of OTUD6B and DDAH2, and a clinically significant mutant form of GNB3.

·         To improve the quality of crystals of citrate synthase and GNB3 and crystallize citrate synthase with novel inhibitors bound to it.

·         To participate in obtaining diffraction data from these protein crystals, either at the Advanced Photon Source or at some other storage-ring source.

·         To participate in analyzing already-determined high-resolution structures to elucidate the role of bound solvent molecules in those structures.

By participating in the experimental project, students will gain experience in protein crystallization, sample preparation for diffraction studies, and collecting and processing diffraction data. By participating in the high-resolution structure redetermination project, students will gain experience in using software for structure analysis and the interaction between macromolecules and solvent.