Core 3

HARC中心：HIV附件和调控复合体 结构生物岩心 摘要 结构生物学核心将开发和优化新的结构生物学方法，使结构 对HARC中心项目重要的重组和重组HIV病毒宿主复合体的测定 和总体目标。我们将把最先进的技术应用于X射线结晶学、单粒子低温 电子显微镜(CRYO-EM)、电子低温断层扫描(CRYO-ET)和抗体(FAB)技术。一个 主要焦点将是开发基于氧化石墨烯(GO)的亲和网格的新功能 技术，并改进基于卷积神经网络(CNN)的成像和体积去噪，以 为低温电磁实验提供更好的图像对齐和分类。我们将开发抗体技术 以促进艾滋病毒-宿主复合体的结构和生物化学表征。其中包括高- 用于深层挖掘重组抗体多样性的吞吐噬菌体抗体珠分选(PHAB分选) 文库，生物层干涉(BLI)免疫吸附分析(BLIISA)，用于筛选和排序候选 结合HITS，以及用于捕获高动态高能态的双半胱氨酸突变体交联法 蛋白质。此外，结构生物学核心是HARC内源性的不可或缺的固定装置 蛋白质结构(HEPS)平台。在与遗传学和蛋白质组学核心的合作下，我们将开发 并使用冷冻-EM方法从感染艾滋病毒的人中纯化内源性标记的宿主蛋白复合体 和未感染的原代CD4+T细胞，用于与结构蛋白质组学数据集成(计算核心) 以生成全面的结构模型。

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.