Rapid structural characterization of most promising novel mAbs and vaccines

结构核心将支持U19中的所有三个项目。U19的目标 研究方案是利用基于结构的合理设计来对抗流感病毒 从治疗性抗体和疫苗的角度来看。这些努力，针对 血凝素（HA）上受体结合位点（RBS）需要多轮设计 并且以迭代方式执行实验验证。因此，这一重点 核心是三方面的：（i）来自项目1的天然抗体将在 与天然HA分子复合。(ii)以解决HA与 来自项目2的工程抗体用于治疗应用，以及（iii）解决 HA工程支架的结构与已知的广泛中和 抗体（bnAbs）在项目3中用于疫苗应用。我们将使用两个电子 显微镜（EM）和X射线晶体学来实现这些目标，并产生 足够的结构覆盖率，以便能够开发抗流感解决方案。 具体来说，我们将使用电子显微镜（EM）来解决复杂的HA结构， 用工程抗体。EM是唯一适合快速生成结构 蛋白质复合物的高通量信息。来自EM的信息 可以直接为进一步的结构和设计工作提供信息。我们将使用两个负 染色和冷冻-EM方法来评估中等和高浓度下抗体-HA复合物 决议，分别。我们还将使用X射线晶体学来解决HA的结构 和用抗体改造的HA支架。X射线晶体学通常用于 揭示蛋白质及其复合物的原子级细节。使用晶体筛选 位于斯克里普斯的结构基因组学联合中心的能力（威尔逊博士是 PI），我们可以筛选大量的抗体、抗原和缀合物复合物。 我们将使用这个管道来解决抗体-HA的原子分辨率结构， 抗体-支架复合物。

The Structure Core will support all three projects in this U19. The goal of our U19 research program is to use structure-based rational design to combat influenza virus from both the therapeutic antibody and vaccine perspective. These endeavors, targeting the receptor-binding site (RBS) on hemagglutinin (HA), require many rounds of design and experimental validation executed in an iterative manner. Thus, the focus of this Core is three-fold: (i) Naturally occurring antibodies from Project 1 will be studied in complex with natural HA molecules. (ii) to solve structures of HA in complex with engineered antibodies from Project 2 for therapeutic applications, and (iii) to solve structures of HA engineered scaffolds in complex with known broadly neutralizing antibodies (bnAbs) in Project 3 for vaccine applications. We will use both electron microscopy (EM) and x-ray crystallography to accomplish these aims and generate sufficient structural coverage to enable development of anti-influenza solutions. Specifically, we will use electron microscopy (EM) to solve structures of HA in complex with engineered antibodies. EM is uniquely suited to rapidly generate structural information of protein complexes in a high-throughput manner. The information from EM can directly inform further structural and design efforts. We will employ both negative stain and cryo-EM methods to evaluate antibody-HA complexes at moderate and high resolution, respectively. We will also use x-ray crystallography to solve structures of HA and engineered HA scaffolds with antibodies. X-ray crystallography is routinely used to reveal atomic-level details of proteins and their complexes. Using the crystal screening capacity of The Joint Center for Structural Genomics housed at Scripps (Dr. Wilson is the PI), we can screen large numbers of antibodies, antigens, and conjugate complexes. We will use this pipeline to solve atomic-resolution structures of antibody-HA and antibody-scaffold complexes.