Core 3

核心3

• 批准号: 10666658
• 负责人: Yifan Cheng
• 金额: $ 70.96万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10666658
• 关键词: Address; Affinity; Antibodies; Bacteriophages; Binding; Biochemical; Biological Assay; Biology; CD4 Positive T Lymphocytes; Cell Extracts; Classification; Collaborations; Complex; Cryoelectron Microscopy; Crystallography; Cysteine; Data; Dependence; Development; Electrons; Genetic; Goals; HIV; HIV Antibodies; Human Cell Line; Image; Immunoglobulin Fragments; Immunosorbents; Integration Host Factors; Interferometry; Libraries; Methodology; Methods; Mining; Mission; Molecular Conformation; Network-based; Oxides; Property; Protein Dynamics; Proteomics; Recombinant Antibody; Recombinants; Research Personnel; Sampling; Sorting; Structural Models; Structure; Technology; Viral; Virus; X-Ray Crystallography; convolutional neural network; crosslink; cryogenics; data integration; denoising; design; electron crystallography; experimental study; flexibility; graphene; image guided; improved; innovation; interest; macromolecule; mutant; novel; particle; protein complex; protein structure; reconstitution; screening; structural biology; technology development; tomography; virus host interaction

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.

HARC中心：HIV配件和监管综合体 结构生物学核心 概括 结构生物学核心将开发和优化新型的结构生物学方法，以实现结构 重组和重组和重组的HIV病毒宿主综合体对HARC中心项目很重要 和整体目标。我们将在X射线晶体学，单个颗粒低温中应用最新技术 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 主要的重点将是基于我们的石墨烯（GO）亲和力网格的新功能的发展 技术，并改善我们的基于卷积神经网络（CNN）的成像和量 提供更好的图像对齐和冷冻EM实验的分类。我们将开发抗体技术 促进HIV宿主复合物的结构和生化表征。这些包括高 吞吐量噬菌体珠珠分选（PHAB排序），用于深化重组AB的多样性 图书馆，生物层干涉法（BLI）免疫吸附测定法（BLIISA）用于筛查和排名 结合命中和一种双半胱氨酸突变体交联方法，用于捕获高能量的高能状态 蛋白质。此外，结构生物学核心是Harc内源性的组成型和必不可少的固定装置 蛋白质结构（HEP）平台。与遗传学和蛋白质组学核心合作，我们将开发 并采用冷冻EM方法进行内源标记的宿主蛋白复合物，从感染的HIV中纯化 和未感染的主要CD4+ T细胞，用于数据集成（计算核），结构蛋白质组学数据 生成全面的结构模型。