膜蛋白的TME3D成像.pptxVIP

3D imaging and quantitative analysis of small solubilized membrane proteins and their complexes By transmission electron microscopy膜蛋白的TME 3D成像胡沁蕊6130111018食品工程Inherently unstable, detergent-solubilized membrane protein complexes can often not be crystallized.背景介绍增溶处理的膜蛋白复合物不稳定，常无法结晶。质量300kDa的，通过冷冻电子显微镜即可获得其三维图像；质量300kDa的，可通过以下两种方法来进行研究：1）扫描透射电镜质量测量；2） 3D -EM 负染样品。实验目的Membrane protein complexes that are too small for cryo-EM and that cannot be crystallized can be quantitatively analyzed by scanning transmission electron microscopy (STEM) and their 3D envelope can be determined by negative stain transmission E M.那些对于冷冻电镜处理来说太小，并且无法结晶的膜蛋白复合物，通过STEM来进行定量分析并且通过透射电镜负染来得到其三维结构。P.S.Membrane proteins fulfill a large variety of vital physiological functions and are important drug targets. Approximately 26% of the human gene shave been estimated to code membrane proteins. Structural information is key to understanding the chemistry and biology of membrane protein function and for developing new drugs. However, the complex physical chemistry of membrane proteins has made them difficult to express, purify and crystallize.实验过程Materials and methods0°实验结果Application of negative staining to aquaporin tetramers45°CTF corrected of（c）（d）颗粒更清楚实验结果3D reconstructionThe resulting structure is elongated along the 4-fold symmetry axis and reflects the missing cone problem. Initial models were generated by applying the standard EMAN2 protocol to class averages calculated from the entire data set including both the 0° and 45° projections.实验结果Imposing the C4 symmetry during model generation yielded a convincing result: the majority of generated models suit for bootstrapping the refinement.3D reconstructionThe refinement was accomplished following the EMAN2 standard protocol, leading to the 3D map 实验结果3D reconstruction3D maps of SoPIP2;1 tetramers solubilized in OG (a), DDM (b), LMNG (c) and AMPH (d). 溶解在不同增溶剂中四聚体的3D图。Maps of DDM (a), LMNG (b) and AMPH (c) solubilized SoPIP2;1 tetramers. 旋转了10°