Structural Biology Shared Resource

PROJECT SUMMARY: STRUCTURAL BIOLOGY SHARED RESOURCE Atomic images of the arrangement of amino acid side chains in three dimensions give the atomic detail needed to visualize the active sites of enzymes, see the DNA-binding sites of transcription factors, and view the protein-protein interactions of signaling molecules. Function can be understood through the determination of atomic structures by X-ray crystallography. Modification of the function of macromolecules is key to developing specific therapies without side effects. In the absence of crystals, the molecular envelopes of macromolecular complexes, individual proteins, and their gross conformational changes upon ligand binding can be determined using small-angle X-ray scattering (SAXS). The SBF (Structural Biology Facility) was initiated many years ago, through funding from the Nebraska Research Initiative (NRI) and the UNMC Vice Chancellor for Research (VCR). Its expanding use by Cancer Center members and its important role in supporting the science of the Cancer Center led to its designation as a Cancer Center core facility beginning in 2008. In the years 2013 through 2015, $1,741,452 was obtained from the NRI and VCR for upgrades. The SBF has four main laboratory services: 1. Protein expression and purification (PrEP). 2. Crystal screening and growth (CSG). 3. X-ray (Small-angle X-ray scattering (SAXS) and single crystal diffraction). 5. Nuclear magnetic resonance (NMR) data collection. The PrEP laboratory provides high-quality purified recombinant protein samples that can be isotopically labeled for NMR data collection, ready for crystallization, or other experiments. The CSG laboratory includes state-of- the-art robotic crystallization instruments with microscale capabilities. SAXS, single-crystal X-ray crystallography, and NMR are used for structure determination. The SBF has two co-directors (one with expertise in protein chemistry and X-ray methods and the other with expertise in NMR) who share the decision making and have experience in structure determination. Two managers, a technician, and one postdoc are employed to develop protein purification and crystallization protocols, maintain the high-tech instrumentation, ensure that the best data is collected/processed, and train users/students.