High Thruput EM Sample Preparation for Structural Biology

用于结构生物学的高通量 EM 样品制备

• 批准号: 8638982
• 负责人: CLINTON S POTTER
• 金额: $ 25.04万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8638982
• 关键词: Address; Area; Automobile Driving; Biological; Biology; Collaborations; Complex; Databases; Electron Microscope; Electrons; G Protein-Coupled Receptor Genes; Goals; Health; Human; Image; Image Analysis; Integral Membrane Protein; Laboratory Research; Manuals; Membrane Proteins; Methodology; Methods; Microfabrication; Miniaturization; Negative Staining; Pattern; Play; Polymers; Preparation; Protein Structure Initiative; Proteins; Reagent; Ribonucleoproteins; Role; Sampling; Solutions; Specimen; Staining method; Stains; Structure; System; T-Cell Activation; Technology; Testing; Transmission Electron Microscopy; Validation; Work; X-Ray Crystallography; base; electron crystallography; experience; improved; instrumentation; macromolecule; member; miniaturize; nanolitre; novel; novel strategies; particle; protein complex; protein structure; public health relevance; research study; skills; structural biology; technology development; transmission process

DESCRIPTION (provided by applicant): There is a scarcity of structures in the Protein Data Bank (PDB) corresponding to integral membrane proteins, higher order eukaryotic proteins, and large protein complexes. Given the pertinence of these classes of proteins to human health, it is clear that novel approaches are still urgently needed to address the challenges posed by these macromolecules that are often related to instability, low yields, and the need to visualize transient complexes. Transmission electron microscopy (TEM) has a role to play in accelerating structure determination in three areas: X-ray crystallography, Electron Crystallography and Single Particle EM (SPEM), but is hampered by low throughput and the sample volume requirements. We proposed to develop a novel approach to TEM specimen preparation, incorporating miniaturization and small volume (picoliter to nanoliter) dispensing that will enable TEM to be integrated seamlessly into the flow of existing high-throughput structural biology efforts. Compared to currently available methodology, our approach dramatically reduces the amount of protein required (1000x) and significantly enhances the overall throughput of TEM sample preparation and imaging (100x). In preliminary work, we have demonstrated that inkjet technology can be utilized to dispense picoliter to nanoliter volumes of reagents with high spatial precision and specimens can be confined to micro- scale regions on a single grid in a defined pattern. We also verified that inkjet dispensing does not disrupt the structure of macromolecules. In specific aim 1, we will develop and test inkjet instrumentation for small volume transfer of specimens from 96-well plates to targeted micro-scale regions on a single EM grid. In specific aim 2, we will use microwell array technology to fabricate novel EM grid substrates capable of segregating ~96 independent samples and incorporate novel materials to provide highly controlled local blotting around each microwell. In specific aim 3, we will integrat these novel EM grids into an automated multi-scale TEM imaging pipeline and test and validate the methods using driving biological projects that are focused on solving structures of integral membrane proteins, low-yield eukaryotic complexes, and transient multi-unit protein assemblies. Members of our team provide the multi-disciplinary experience and skills (instrumentation and microfabrication, structural biology, automated TEM) required to accomplish all aspects of this project. We have strong support from members of the PSI: Biology Network who are enthusiastically supportive of the potential for using an HT-TEM platform to accelerate structure determination of challenging macromolecular systems.

描述（由申请人提供）：蛋白质数据库（PDB）中的结构稀缺，与整体膜蛋白，高阶真核蛋白和大蛋白质复合物相对应。鉴于这些类别蛋白质与人类健康的相关性，很明显，仍然需要急需新的方法来应对这些大分子通常与不稳定性，低产量和可视化瞬态复合物相关的挑战。透射电子显微镜（TEM）在三个领域的加速结构测定中起作用：X射线晶体学，电子晶体学和单个粒子EM（SPEM），但受到低吞吐量和样品体积需求的阻碍。我们建议开发一种新型的TEM标本制备方法，结合微型化和小体积（纳米尺的Picoliter），以启用 将无缝集成到现有高通量结构生物学工作的流动中。与当前可用的方法相比，我们的方法大大减少了所需蛋白质的量（1000倍），并显着增强了TEM样品制备和成像的总体吞吐量（100x）。在初步工作中，我们证明了喷墨技术可用于将picoliter分配给纳米仪的纳米材料量，这些试剂量很高。 精度和样品可以局限于定义模式的单个网格上的微观区域。我们还验证了喷墨分配不会破坏大分子的结构。在特定的目标1中，我们将开发和测试喷墨仪器，以将标本从96孔板的小体积转移到单个EM网格上的靶向微型区域。在特定的目标2中，我们将使用Microwell阵列技术来制造能够隔离〜96个独立样品的新型EM网格底物，并结合新型材料，以提供每个微孔周围高度控制的局部印迹。在特定的目标3中，我们将将这些新型EM网格集成到自动的多尺度TEM成像管道中，并使用驱动生物学项目进行测试并验证方法，这些项目专注于求解整体膜蛋白的结构，低覆盖型真核化合物复合物，瞬态多核蛋白质和瞬态多单位蛋白质组件。我们团队的成员提供了完成该项目的所有方面所需的多学科经验和技能（仪器和微分化，结构生物学，自动化TEM）。我们得到了PSI：生物学网络成员的大力支持，他们热情地支持使用HT-TEM平台加速挑战性大分子系统的结构的潜力。