An automated pipeline to screen membrane protein 2D crystallization.
An automated pipeline to screen membrane protein 2D crystallization.
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DOI：
10.1007/s10969-010-9088-5
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发表时间：
2010-06
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影响因子：
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通讯作者：
Ubarretxena-Belandia, Iban
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文献类型：
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作者：
Kim, Changki;Vink, Martin;Hu, Minghui;Love, James;Stokes, David L;Ubarretxena-Belandia, Iban
电子晶体学依赖于二维（2D）晶体的电子低温显微镜，特别适合于研究膜蛋白在其天然脂质双层环境中的结构。为了从纯化的膜蛋白获得2D晶体，蛋白质-脂质-去污剂三元混合物中的去污剂必须在有利于重构成蛋白脂质体和形成有序晶格的条件下去除，通常通过透析。为了确定这些条件下的广泛的参数，如pH值，脂质组成，脂质与蛋白质的比例，离子强度和配体必须在一个程序中进行筛选，涉及四个步骤：结晶，样品制备电子显微镜，图像采集和评价。传统上，这些步骤是手动进行的，因此，2D结晶试验的范围受到限制。因此，我们开发了一种自动化的流水线来筛选2D晶体的形成。我们采用了96孔透析块，在广泛的条件下重建的目标蛋白，旨在促进结晶。使用96位磁性平台和液体处理机器人并行制备阴性染色标本。将自动化滤线栅插入电子显微镜和计算机化图像采集确保了对结晶屏的快速评估。迄今为止，已经对15种不同的膜蛋白进行了38次二维结晶筛选，总计超过3000次单独的结晶实验。这些蛋白质中有三种已经产生了衍射2D晶体。我们的自动化流水线在产量和再现性方面优于传统的2D结晶方法。
Electron crystallography relies on electron cryomicroscopy of two-dimensional (2D) crystals and is particularly well suited for studying the structure of membrane proteins in their native lipid bilayer environment. To obtain 2D crystals from purified membrane proteins, the detergent in a protein-lipid-detergent ternary mixture must be removed, generally by dialysis, under conditions favoring reconstitution into proteoliposomes and formation of well-ordered lattices. To identify these conditions a wide range of parameters such as pH, lipid composition, lipid-to-protein ratio, ionic strength and ligands must be screened in a procedure involving four steps: crystallization, specimen preparation for electron microscopy, image acquisition, and evaluation. Traditionally, these steps have been carried out manually and, as a result, the scope of 2D crystallization trials has been limited. We have therefore developed an automated pipeline to screen the formation of 2D crystals. We employed a 96-well dialysis block for reconstitution of the target protein over a wide range of conditions designed to promote crystallization. A 96-position magnetic platform and a liquid handling robot were used to prepare negatively stained specimens in parallel. Robotic grid insertion into the electron microscope and computerized image acquisition ensures rapid evaluation of the crystallization screen. To date, 38 2D crystallization screens have been conducted for 15 different membrane proteins, totaling over 3000 individual crystallization experiments. Three of these proteins have yielded diffracting 2D crystals. Our automated pipeline outperforms traditional 2D crystallization methods in terms of throughput and reproducibility.