Technology Development 1: Development of nanocrystal processing protocols for microED

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Abstract for Technology Development Program 1 - MicroED X-ray crystallography over the last 50 years has been the most successful method to obtain atomic structures of biological molecules. Crystallization of large multi-protein complexes is challenging, and even if successful, the resulting crystals are usually small, delicate and present multiple challenges. New developments such as serial femtosecond crystallography using a free electron laser (FEL) and electron crystallography or micro-electron diffraction (microED) are alternative approaches to obtain structures, using nano-meter or low micro-meter sized crystals (nanocrystals). Solving structures of bio-macromolecules using FELs requires billions of nanocrystals, amounts of material that frequently is not available. Solving structures with microED, on the other hand, may, in principle, be possible with only a few nanocrystals, thus overcoming problems related to sample quantity. The Calero laboratory at the University of Pittsburgh has pioneered nanocrystal discovery and optimization using transmission electron microscopy (TEM). This has permitted to significantly increase the number of potential crystallization conditions through direct observation of crystal lattices. In this technology development proposal, we intend to develop new methodologies to determine structures of protein complexes of HIV-1 proteins and their human binding partners by exploring nano crystallization space and optimizing stabilizing conditions for the application of microED approaches for structure determination.