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: 8811451
• 负责人: CLINTON S POTTER
• 金额: $ 35.86万
• 依托单位: NEW YORK STRUCTURAL BIOLOGY CENTER
• 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 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8811451
• 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; 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）：在蛋白质数据库（PDB）中缺乏与完整膜蛋白、高级真核蛋白和大蛋白复合物相对应的结构。鉴于这些类别的蛋白质与人类健康的相关性，很明显，仍然迫切需要新的方法来解决这些大分子所带来的挑战，这些大分子通常与不稳定性，低产量和可视化瞬时复合物的需求有关。透射电子显微镜（TEM）在加速三个领域的结构测定中发挥作用：X射线晶体学，电子晶体学和单粒子EM（SPEM），但受到低通量和样品体积要求的阻碍。我们建议开发一种新的TEM样品制备方法，将小型化和小体积（皮升到纳升）点胶结合起来， TEM将无缝集成到现有的高通量结构生物学工作流程中。与目前可用的方法相比，我们的方法大大减少了所需的蛋白质量（1000倍），并显着提高了TEM样品制备和成像的整体通量（100倍）。在初步工作中，我们已经证明了喷墨技术可以用于以高空间分辨率分配皮升到纳升体积的试剂。 精确度和样本可以以定义的模式限制在单个网格上的微观区域。我们还验证了喷墨点胶不会破坏大分子的结构。在具体目标1中，我们将开发和测试喷墨仪器，用于将样品从96孔板小体积转移到单个EM网格上的目标微尺度区域。在具体目标2中，我们将使用微孔阵列技术制造能够分离约96个独立样品的新型EM网格基底，并结合新型材料以在每个微孔周围提供高度受控的局部印迹。在具体目标3中，我们将把这些新的EM网格集成到自动化的多尺度TEM成像管道中，并使用驱动生物项目来测试和验证这些方法，这些生物项目专注于解决完整膜蛋白、低产量真核复合物和瞬时多单元蛋白质组装体的结构。我们团队的成员提供完成该项目各个方面所需的多学科经验和技能（仪器和微加工，结构生物学，自动TEM）。我们得到了PSI：Biology Network成员的大力支持，他们热情地支持使用HT-TEM平台来加速具有挑战性的大分子系统的结构测定。