High-throughput Pipeline for Electron Crystallography

电子晶体学高通量管道

• 批准号: 8313999
• 负责人: David L. Stokes
• 金额: $ 29.7万
• 依托单位: NEW YORK STRUCTURAL BIOLOGY CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8313999
• 关键词: Algorithms; Back; Behavior; Biological; Biology; Cell Adhesion Molecules; Cell physiology; Cells; Computer software; Cryoelectron Microscopy; Crystallization; Crystallography; Cyclodextrins; Data; Data Collection; Databases; Detergents; Development; Dialysis procedure; Disease; Drug Design; Electron Microscope; Electrons; Elements; Environment; Enzymes; Excision; Freezing; Image; Licensing; Lipids; Management Information Systems; Mediating; Membrane; Membrane Proteins; Methods; Microfluidic Microchips; Microfluidics; Modeling; NMR Spectroscopy; Outcome; Pharmaceutical Preparations; Process; Proteins; Proteome; Public Health; Resolution; Robot; Robotics; Roentgen Rays; Sampling; Screening procedure; Shapes; Signal Transduction; Solutions; Staining method; Stains; Structural Protein; Structure; Testing; Text; Therapeutic; United States; Urticaria; Visit; X-Ray Crystallography; data acquisition; density; design; electron crystallography; novel; operation; programs; protein structure; public health relevance; receptor; reconstitution; tool; two-dimensional

DESCRIPTION (provided by applicant): Biological membranes surround all cells and mediate their interactions with the outside world. Depending on the biological context, membrane proteins act as receptors, enzymes, channels, transporters, structural proteins and cell adhesion molecules and, as such, contribute to a wide variety of essential cellular functions. Structural information for membrane proteins is relatively scarce, despite the fact that they represent the target of 60% of therapeutic drugs sold in the United States. We propose to establish a pipeline for determining membrane protein structures by electron crystallography, which is the application of cryo-electron microscopy to two-dimensional crystals. To prepare such crystals, the membrane bilayer is reconstituted with a high density of purified membrane proteins, thus providing a native membrane environment and fewer crystallization constraints for the constituent proteins. Electron crystallography has an established track record in producing structures at both atomic and intermediate resolutions and represents a valuable alternative to X-ray crystallography and NMR spectroscopy, which are generally constrained to studying detergent-solubilized species. To date, electron crystallography remains a low-throughput operation, which has significantly reduced its contribution to membrane protein biology. We have developed some tools to overcome the bottlenecks in screening crystallization conditions, which we seek to expand in the current application. Specifically, we propose further developments for crystallization on a 96-well format by implementing a microfluidic device for dialysis that minimizes sample volumes and by using cyclodextrins to control detergent removal rates in an effort to optimize crystal quality. By studying a wide range of different target proteins, we will empirically establish factors that are most critical to influencing the crystallization process and develop a set of conditions that are generally effective for screening new proteins with unknown crystallization behaviors. We will continue developing our methods for robotic imaging of crystallization screens. The process of preparing samples and imaging them within the electron microscope currently represents the most significant bottleneck limiting the number of conditions that can be explored. We have built a robot for sample insertion and have interfaced it with automated image acquisition software, but we propose to add shape recognition to this software to maximize its efficiency. We will integrate the resulting images within an established LIMS database for keeping track of the structure determination pipeline and will implement shape recognition to enable automated assignment of scores to crystallization trials. Finally, we propose to develop an application for high resolution data collection from well-ordered crystals, thus facilitating the optimization of crystal size and order and, ultimately, acquisition of the data required for structure determination. We are convinced that by applying high- throughput methods to 2D crystallization and image acquisition, electron crystallography can make a substantial contribution to our understanding of membrane protein biology. PUBLIC HEALTH RELEVANCE: Membranes surround all cells and proteins within these membrane mediate the flow of information and materials. As a result, membrane proteins are implicated in many diseases. Structural information about these proteins is critical to understanding the biology behind the disease and for designing drugs to ameliorate the problems.

描述(申请人提供)：生物膜包围所有细胞，并调节它们与外界的相互作用。根据生物环境的不同，膜蛋白作为受体、酶、通道、转运体、结构蛋白和细胞黏附分子发挥作用，因此有助于发挥多种基本的细胞功能。膜蛋白的结构信息相对稀少，尽管它们代表了在美国销售的60%的治疗药物的靶标。我们建议建立一种用电子结晶学来确定膜蛋白质结构的管道，这是冷冻电子显微镜在二维晶体中的应用。为了制备这种晶体，膜双层与高密度的纯化膜蛋白重组，从而为组成蛋白提供了一个自然的膜环境和较少的结晶限制。电子结晶学在产生原子和中间分辨率的结构方面有着既定的记录，是X射线结晶学和核磁共振光谱的有价值的替代方法，后者通常仅限于研究洗涤剂增溶物种。到目前为止，电子结晶学仍然是一种低通量的操作，这大大减少了它对膜蛋白生物学的贡献。我们已经开发了一些工具来克服筛选结晶条件的瓶颈，我们希望在当前的应用中扩大这一点。具体地说，我们建议在96孔格式上进一步开发结晶，通过实施微流控设备来最大限度地减少样品体积，并使用环糊精来控制洗涤剂的去除速度，以努力优化晶体质量。通过研究一系列不同的目标蛋白质，我们将经验地确定对结晶过程最关键的因素，并开发一套普遍有效的条件，用于筛选具有未知结晶行为的新蛋白质。我们将继续开发用于结晶屏幕的机器人成像的方法。制备样品并在电子显微镜中对其成像的过程目前是限制可以探索的条件数量的最大瓶颈。我们已经建造了一个用于样品插入的机器人，并将其与自动图像采集软件进行了接口，但我们建议在该软件中添加形状识别功能，以最大限度地提高其效率。我们将把生成的图像整合到一个已建立的LIMS数据库中，以跟踪结构确定管道，并将实施形状识别，以实现结晶试验的自动评分。最后，我们建议开发一个从有序晶体中收集高分辨率数据的应用程序，从而促进晶体尺寸和顺序的优化，并最终获得结构确定所需的数据。我们相信，通过将高通量方法应用于2D结晶和图像采集，电子结晶学可以为我们理解膜蛋白生物学做出实质性贡献。 与公共卫生相关：膜包围着所有细胞，这些膜内的蛋白质调节信息和材料的流动。因此，膜蛋白与许多疾病有关。关于这些蛋白质的结构信息对于理解疾病背后的生物学和设计改善问题的药物至关重要。