Electron Microscopy of Membrane Proteins

描述(申请人提供)：膜蛋白约占生物体基因产物的25%。它们分布在细胞膜和细胞器膜上，对于跨越这些疏水隔膜的通讯是必不可少的。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，一些膜蛋白转运蛋白的结构和作用机制将用冷冻电子显微镜和图像分析来研究。来自ATP结合盒(ABC)、主要促进剂(MFS)和多抗菌毒素挤出(Mate)超家族的转运蛋白将被研究，因为这些是目前由一名研究人员平行进行的X射线结晶学研究的重点。ABC、MFS和Mate超级家族的成员具有非常重要的医学意义，因为他们对抗生素和化疗耐药负有责任。 冷冻-EM和图像分析将被用来计算(I)螺旋管和2D晶体的MSBA-ABC转运体-在不同的核苷酸结合和底物结合的状态下的3D MAP，以及(Ii)FucP-MFS转运体的2D晶体的底物结合的3D MAP。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和运输机制。 其他实验将集中在筛选结晶条件，以改善现有螺旋和2D阵列的有序性，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.