ELECTRON MICROSCOPY OF MEMBRANE PROTEINS(RMI)

描述（申请人提供）：膜蛋白约占生物体基因产物的25%。它们占据细胞和细胞器膜，并且对于跨越这些疏水性隔室边界的通信是必不可少的。它们参与识别、运输和转导过程，这些过程是生命系统的基本属性。 在这里描述的工作中，将通过冷冻电子显微镜和图像分析来研究一些膜蛋白转运蛋白的结构和作用机制。将研究来自ATP结合盒（ABC）、主要易化因子（MFS）和多抗菌毒素挤出（MATE）超家族的转运蛋白，因为这些是其中一位研究者目前平行X射线晶体学研究的重点。ABC、MFS和MATE超家族的成员具有重要的医学意义，因为它们负责抗生素和化疗耐药性。 Cryo-EM和图像分析将用于计算（i）各种核苷酸结合和底物结合状态下MsbA（ABC转运蛋白）的螺旋管和2D晶体以及（ii）底物结合的FucP（MFS转运蛋白）的2D晶体的3D图谱。这些图谱将揭示这两个膜蛋白超家族的代表性成员的结构，并提供对转运机制的见解。 额外的实验将集中在筛选结晶条件，以改善现有的螺旋和二维阵列的顺序，并为EM结构研究生长其他膜蛋白的有序阵列。

DESCRIPTION (provided by applicant): Membrane proteins account for approximately 25% of gene products in organisms. They populate cell and organelle membranes and are essential for communication across these hydrophobic compartmental boundaries. They participate in recognition, transport and transduction processes that are fundamental attributes of living systems. In the work described here, the structure and mechanism of action of a number of membrane protein transporters will be investigated by cryo-electron microscopy and image analysis. Transporters from the ATP Binding Cassette (ABC), Major Facilitator (MFS) and Multi Antimicrobial Toxin Extrusion (MATE) superfamilies will be studied, as these are the current focus of parallel x-ray crystallographic investigations by one of the investigators. Members of the ABC, MFS and MATE superfamilies are of great medical importance as they are responsible for antibiotic and chemotherapy resistance. Cryo-EM and image analysis will be used to calculate 3D maps from (i) helical tubes and 2D crystals of MsbA - an ABC transporter - in various nucleotide-bound and substrate-bound states and, (ii) 2D crystals of FucP - an MFS transporter - with substrate bound. The maps will reveal the structures and provide insights into the transport mechanisms of representative members of these two membrane protein superfamilies. Additional experiments will focus on screening crystallization conditions to improve the order of the existing helical and 2D arrays, and to grow ordered arrays of other membrane proteins for EM structural studies.