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: 8996767
• 负责人: CLINTON S POTTER
• 金额: $ 12.74万
• 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/8996767
• 关键词: Thruput; EM; Sample; Preparation; Structural

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）中缺少与完整膜蛋白、高阶真核蛋白和大蛋白复合物相对应的结构。鉴于这类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、低产量和可视化瞬态复合物的需要有关。透射电子显微镜（TEM）在加速x射线晶体学、电子晶体学和单粒子电子显微镜（SPEM）三个领域的结构测定方面发挥着重要作用，但由于低通量和样品体积要求而受到阻碍。我们建议开发一种新的TEM样品制备方法，结合小型化和小体积（皮升到纳升）分配，这将使