Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy.
Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy.
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作者：
结构生物学中一个长期追求的目标是在其膜环境中对膜蛋白进行成像。这一目标已经实现了电子晶体学在这些特殊情况下，蛋白质形成高度有序的阵列在脂质双层。它也已经实现了通过NMR方法在蛋白质高达50千道尔顿（kDa）的大小，虽然毫克量的蛋白质和同位素标记是必需的。对于微克量的大的可溶性蛋白质的结构分析，一个越来越强大的方法，不需要结晶是从低温冷却样品的电子显微镜（电子低温显微镜（cryo-EM））的单颗粒重建。在这里，我们报告的第一个单粒子冷冻电镜研究的膜蛋白，人类大电导钙和电压激活钾通道（BK），在脂质环境中。这种新方法被称为随机球约束（RSC）单粒子重建。BK通道，六跨膜段（6 TM）离子通道家族的成员，在低密度下重组成脂质囊泡（脂质体），并通过钾通量测定验证其功能。囊泡也被冷冻在玻璃冰中，并在电子显微镜下成像。从8，400个单个蛋白质颗粒的图像中，以1.7-2.0 nm的分辨率获得了BK通道及其膜环境的三维（3D）重建。由于不需要形成晶体，RSC方法有望在许多其他膜蛋白的结构研究中也很有用。
A long-sought goal in structural biology has been the imaging of membrane proteins in their membrane environments. This goal has been achieved with electron crystallography in those special cases where a protein forms highly ordered arrays in lipid bilayers. It has also been achieved by NMR methods in proteins up to 50 kilodaltons (kDa) in size, although milligram quantities of protein and isotopic labelling are required. For structural analysis of large soluble proteins in microgram quantities, an increasingly powerful method that does not require crystallization is single-particle reconstruction from electron microscopy of cryogenically cooled samples (electron cryomicroscopy (cryo-EM)). Here we report the first single-particle cryo-EM study of a membrane protein, the human large-conductance calcium- and voltage-activated potassium channel (BK), in a lipid environment. The new method is called random spherically constrained (RSC) single-particle reconstruction. BK channels, members of the six-transmembrane-segment (6TM) ion channel family, were reconstituted at low density into lipid vesicles (liposomes), and their function was verified by a potassium flux assay. Vesicles were also frozen in vitreous ice and imaged in an electron microscope. From images of 8,400 individual protein particles, a three-dimensional (3D) reconstruction of the BK channel and its membrane environment was obtained at a resolution of 1.7–2.0 nm. Not requiring the formation of crystals, the RSC approach promises to be useful in the structural study of many other membrane proteins as well.
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DOI：
10.1073/pnas.0711533105
发表时间：
2008-02-05
影响因子：
11.1
作者：
Clayton, Gina M.;Altieri, Steve;Morais-Cabral, Joao H.
通讯作者：
Morais-Cabral, Joao H.
影响因子：
64.8
作者：
Long, Stephen B.;Tao, Xiao;MacKinnon, Roderick
通讯作者：
MacKinnon, Roderick
DOI：
10.1085/jgp.200509419
发表时间：
2006-06
期刊：
The Journal of general physiology
影响因子：
--
作者：
Fodor AA;Aldrich RW
通讯作者：
Aldrich RW
影响因子：
5.6
作者：
GRIGORIEFF, N;BECKMANN, E;ZEMLIN, F
通讯作者：
ZEMLIN, F
影响因子：
16.8
作者：
Yang, Huanghe;Shi, Jingyi;Zhang, Guohui;Yang, Junqiu;Delaloye, Kelli;Cui, Jianmin
通讯作者：
Cui, Jianmin