Core 3

THE HARC CENTER: HIV ACCESSORY AND REGULATORY COMPLEXES STRUCTURAL BIOLOGY CORE SUMMARY The Structural Biology Core will develop and optimize novel structural biology approaches that enable structure determination of recombinant and reconstituted HIV virus-host complexes important to HARC Center Projects and overall goals. We will apply state-of-the-art technologies in X-ray crystallography, single particle cryogenic electron microscopy (cryo-EM), electron cryogenic tomography (cryo-ET), and antibody (Fab) technologies. A major focus will be the development of novel functionalities for our graphene oxide (GO) based affinity grids technology, and improving our convolutional neural network (CNN) based imaging and volume denoising to provide better image alignment and classification of cryo-EM experiments. We will develop antibody technologies to facilitate the structural and biochemical characterization of HIV-host complexes. These include high- throughput Phage Antibody Bead Sorting (PhAB Sorting) for deep mining of the diversity of recombinant Ab libraries, Bio-layer Interferometry (BLI) Immunosorbent Assay (BLIISA) for screening and ranking of candidate binding hits, and a double cysteine mutant cross-linking method for trapping high energy states of highly dynamic proteins. Furthermore, the Structural Biology Core is an integral and essential fixture of the HARC endogenous protein structure (HEPS) platform. In collaboration with the Genetics and Proteomics Cores, we will develop and employ cryo-EM approaches for endogenously tagged host protein complexes purified from HIV infected and uninfected primary CD4+ T cells for data integration (Computational Core) with structural proteomics data to generate comprehensive structural models.