Determine high-resolution structure of membrane protein by single particle cryoEM

描述（由申请人提供）：膜蛋白在许多细胞过程中起关键作用。膜蛋白的高分辨率结构为理解其功能机制提供了关键。然而，确定膜蛋白的高分辨率结构是特别困难和具有挑战性的。除了膜蛋白表达和纯化之外，X射线晶体学中的关键障碍之一是结晶步骤。在这项提案中，我们将开发一种基于单粒子电子冷冻显微镜（cryoEM）的创新技术，以确定亚纳米或更高分辨率的膜蛋白结构。在这种方法中，膜蛋白在脂质环境中保持其天然构象，而不需要形成晶体。单颗粒cryoEM已成为研究可溶性蛋白质复合物结构的通用工具，而无需大量蛋白质和形成晶体。它在研究具有二十面体对称性的病毒方面实现了原子分辨率，在研究没有高度对称性的行为良好的大蛋白质复合物方面实现了近原子分辨率。然而，应用这种方法来确定膜蛋白结构，以类似的分辨率是不简单的。它需要创新的方法来克服许多技术难题，例如如何在cryoEM中成像非常小的膜蛋白，以及如何精确地计算对齐非常嘈杂的膜蛋白图像。我们提出了一种新的方法来克服这些困难，并使高分辨率的结构测定膜蛋白的单粒子cryoEM。为了测试我们的新方法，我们将确定两个重要的膜蛋白的结构：哺乳动物囊泡谷氨酸转运（VGLUT）和氯喹抗性转运蛋白（PfCRT）的细菌同系物。VGLUTs通过胞吐作用将谷氨酸转运到突触囊泡中进行调节释放，从而在兴奋性神经传递中发挥基础作用。PfCRT在恶性疟原虫对喹啉类抗疟药的耐药性中起关键作用。我们的目标是确定这两种蛋白质的结构，以亚纳米分辨率，并最终接近原子分辨率。我们还将简化我们的方法，以便它可以应用于许多其他膜蛋白。

DESCRIPTION (provided by applicant): Membrane proteins play critical roles in many cellular processes. High-resolution structures of membrane proteins provide keys to understand the mechanism of their functions. However, it is particularly difficult and challenging to determine high-resolution structure of membrane protein. One of the critical barriers in X-ray crystallography, in addition to membrane protein expression and purification, is at the step of crystallization. In this proposal, we will develop an innovative technology based on single particle electron cryo-microscopy (cryoEM) to determine structure of membrane proteins to subnanometer or higher resolution. In this method, membrane proteins are kept in their native conformation within lipid environment without need to form crystals. Single particle cryoEM has become a versatile tool in studying structures of soluble protein complexes without need of a large amount of proteins and forming crystals. It has achieved atomic resolution in studying viruses with icosahedral symmetry and near atomic resolution of well-behaved large protein complexes without high symmetry. However, applying this method to determine membrane protein structures to similar resolution is not straightforward. It requires innovative approach to overcome many technical difficulties, such as how to image very small membrane proteins in cryoEM and how to computationally align very noisy images of membrane proteins accurately. We proposed a novel approach to overcome these difficulties and to enable high-resolution structure determination of membrane proteins by single particle cryoEM. To test our novel approach, we will determine structures of two important membrane proteins: a bacterial homologue of mammalian vesicular glutamate transport (VGLUTs) and a Chloroquine resistance transporter (PfCRT). The VGLUTs transport glutamate into synaptic vesicle for regulated release by exocytosis, thus play a fundamental role in excitatory neurotransmission. The PfCRT plays a critical role in Plasmodium falciparum resistance to the quinoline antimalarials. We aim to determine the structure of these two proteins to subnanometer resolution and eventually to near atomic resolution. We will also streamline our approach so that it can be applied to many other membrane proteins.