Cryo-EM structure of a membrane-embedded glutamate receptor

描述(申请人提供)：结构生物学的一个长期追求的目标是确定膜蛋白在其天然膜环境中的结构，以及由底物分子和膜电位的跨膜梯度所定义的各种功能状态的结构。在X射线结构测定的情况下，应用这种梯度是不可能的，更不用说在膜环境中结晶了。对于2D晶体的电子结晶学分析，梯度的应用也是遥不可及的。建议的工作是进一步发展一种通用的方法，用于确定重组为脂质体的膜蛋白的单颗粒冷冻-EM结构。这种方法被称为随机球约束(RSC)单粒子重建。在拟议的工作中，我们将努力克服几个剩余的技术障碍，然后将生物化学、EM成像和图像处理方法的组合应用于GluA2谷氨酸受体的纳米分辨率结构，将其重组为脂膜。

DESCRIPTION (provided by applicant): A long-pursued goal of structural biology has been the determination of the structure of membrane proteins in their native membrane environment, and in their various functional states as defined by transmembrane gradients of substrate molecules and membrane potentials. Application of such gradients, let alone crystallization in a membrane environment, has not been possible in the case of X-ray structure determination. The application of gradients has remained out of reach as well for the analysis of 2D crystals by electron crystallography. The proposed work concerns the further development of a general method for single-particle cryo-EM structure determination of membrane proteins reconstituted into liposomes. The method is called random spherically- constrained (RSC) single-particle reconstruction. In the proposed work we will work to surmount several remaining technical hurdles, and then apply this combination of biochemistry, EM imaging and image processing methods toward a nanometer- resolution structure of the GluA2 glutamate receptor as reconstituted into a lipid membrane.