High Thruput EM Sample Preparation for Structural Biology

描述(申请人提供)：蛋白质数据库(PDB)中缺少与完整膜蛋白、高阶真核蛋白质和大型蛋白质复合体相对应的结构。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带来的挑战，这些挑战往往与不稳定、低产率和可视化瞬时复合体的需要有关。在X射线结晶学、电子结晶学和单粒子电子显微镜(SPEM)三个领域中，透射式电子显微镜(TEM)在加速结构确定方面发挥了作用，但由于吞吐量低和样品量要求较高而受到限制。我们建议开发一种新的透射电子显微镜样品制备方法，结合小型化和小体积(皮升到纳升)的分配，这将使 TEM将无缝地整合到现有的高通量结构生物学努力的流中。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数量(1000倍)，并显著提高了透射电子显微镜样品制备和成像的总体吞吐量(100倍)。在初步工作中，我们已经证明了喷墨技术可以用来分配皮升到纳升体积的高空间试剂。 精度和样本可以被限制在定义图案的单个网格上的微尺度区域。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来制备能够分离~96个独立样品的新型EM栅格基板，并结合新材料在每个微孔周围提供高度可控的局部印迹。在具体目标3中，我们将把这些新颖的EM网格整合到一个自动化的多尺度透射电子显微镜成像管道中，并使用驱动生物项目来测试和验证这些方法，这些项目专注于解决完整膜蛋白、低产率真核复合体和瞬时多单位蛋白质组装的结构。我们团队的成员提供了完成该项目所有方面所需的多学科经验和技能(仪器和微制造、结构生物学、自动透射电子显微镜)。我们得到了PSI：生物网络成员的大力支持，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确定的潜力。

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.