Chemically engineered bilayers for cryoEM imaging of membrane proteins in continuous membranes

细胞通过膜蛋白与环境相互作用。结构和功能研究 因此，膜蛋白非常重要。真核细胞膜蛋白的结构测定 然而，尽管取得了实质性进展，但膜仍然很困难。部分挑战来自于 许多真核细胞膜蛋白经历复杂的细胞内成熟过程， 在到达最终目的地之前进行不同的翻译后修饰。当前高通量 结晶和cryoEM单颗粒重建主要是用洗涤剂中的蛋白质进行的，或者 膜模拟系统，如双胞、纳米盘、脂质立方相或Amphipol，其中仍然存在 与天然膜相比有显著差异。需要新技术来克服 这些问题我们在这里提出了两个新的技术，冷冻电镜研究膜蛋白 在连续膜中使用1型IP 3受体（IP 3R）作为工作模型。这两个前提 方法部分是基于我们最近的工作，化学工程程序，适用于 功能化纳米厚碳膜和珠支撑的球形单层膜（bSUM） 系统，允许产生稳定的巨大单层囊泡。使用毫克量的IP 3R蛋白， 我们将制备纳米bSUM（nm-bSUM）和碳支撑平面单层膜（cPUM）。 这两个系统将准备用于连续膜中IP 3R的cryoEM可视化， 蛋白质完全浸入脂质双层中，使我们能够从图像中解析受体结构。 膜整合分子的能力。nm-bSUM中受体的图像将用于随机球形分析。 约束（RSC）重建。将在高倾斜角度下对cells中的受体进行成像，以进行3D重建 并对成像场上散焦水平的变化进行校正。这两种方法都将依赖于化学 工程和膜重建在纳米尺度上，并将导致有效的单向 在亚nM浓度下插入膜蛋白，这将特别有利于选择 特异性标记的成熟功能性膜蛋白或富集低丰度膜蛋白 在亚nM浓度下的复合物。拟议研究的结果将创造新的机会之窗 用于膜中各种膜蛋白复合物的冷冻-EM研究和用于使用纳米级膜 其他生物分析或生物医学应用中的系统。 !一个！

Cells interact with their environments through membrane proteins. Structural and functional studies of membrane proteins are thus very important. Structure determination of eukaryotic membrane proteins in membrane however remains difficult despite substantial progresses. Part of the challenge comes from the fact that many eukaryotic membrane proteins undergo a complicated intracellular maturation process and carry different post-translational modifications before reaching their final destinations. Current high throughput crystallization and cryoEM single particle reconstruction are largely carried out with proteins in detergents, or in membrane-mimetic systems such as bicelles, nanodiscs, lipid-cubic phases or amphipols, where there are still significant differences in comparison with a native membrane. New technologies are needed to overcome these problems. We propose here to develop two new technologies for cryoEM study of membranes proteins in continuous membrane using type 1 IP3 receptor (IP3R) as a working model. The premise of these two methods is partly based on our recent work of a chemical engineering procedure that is suitable for functionalizing nanometer-thick carbon films and of a bead-supported spherical unilamellar membrane (bSUM) system that allows the generation of stable giant unilamellar vesicles. With milligram amounts of IP3R proteins, we will produce a nanometer-bSUM (nm-bSUM) and a carbon-supported planar unilamellar membrane (cPUM). These two systems will be prepared for the cryoEM visualization of the IP3Rs in continuous membrane where the proteins are fully immersed in a lipid bilayer, and will allow us to resolve the receptor structure from images of membrane-integrated molecules. Images of the receptors in nm-bSUMs will be used for random spherically constrained (RSC) reconstruction. Receptors in cPUMs will be imaged at high tilt angles for 3D reconstruction with corrections for changes in defocus levels across the imaging field. Both methods will rely on chemical engineering and membrane reconstitution at the nanometer scale and will result in efficient unidirectional insertion of membrane proteins at sub-nM concentrations, which will be particularly beneficial for selecting specifically labeled mature functional membrane proteins or enriching low-abundance membrane protein complexes at sub-nM concentrations. Results of the proposed studies will create new windows of opportunities for cryo-EM study of various membrane protein complexes in membrane and for using nanoscale membrane systems in other bioanalytical or biomedical applications. ! 1!