New York Structural Biology Center, New York, NY 10027, USA.
J Struct Biol. 2010 Jul;171(1):102-10. doi: 10.1016/j.jsb.2010.02.018. Epub 2010 Mar 1.
Membrane proteins fulfill many important roles in the cell and represent the target for a large number of therapeutic drugs. Although structure determination of membrane proteins has become a major priority, it has proven to be technically challenging. Electron microscopy of two-dimensional (2D) crystals has the advantage of visualizing membrane proteins in their natural lipidic environment, but has been underutilized in recent structural genomics efforts. To improve the general applicability of electron crystallography, high-throughput methods are needed for screening large numbers of conditions for 2D crystallization, thereby increasing the chances of obtaining well ordered crystals and thus achieving atomic resolution. Previous reports describe devices for growing 2D crystals on a 96-well format. The current report describes a system for automated imaging of these screens with an electron microscope. Samples are inserted with a two-part robot: a SCARA robot for loading samples into the microscope holder, and a Cartesian robot for placing the holder into the electron microscope. A standard JEOL 1230 electron microscope was used, though a new tip was designed for the holder and a toggle switch controlling the airlock was rewired to allow robot control. A computer program for controlling the robots was integrated with the Leginon program, which provides a module for automated imaging of individual samples. The resulting images are uploaded into the Sesame laboratory information management system database where they are associated with other data relevant to the crystallization screen.
膜蛋白在细胞中发挥着许多重要作用，是大量治疗药物的作用靶点。尽管膜蛋白的结构测定已成为当务之急，但事实证明这在技术上具有挑战性。二维（2D）晶体的电子显微镜具有在其天然脂质环境中可视化膜蛋白的优势，但在最近的结构基因组学努力中未得到充分利用。为了提高电子晶体学的普遍适用性，需要高通量方法来筛选大量的 2D 结晶条件，从而增加获得有序晶体的机会，从而实现原子分辨率。以前的报告描述了用于在 96 孔格式上生长 2D 晶体的设备。本报告描述了一种用于使用电子显微镜自动对这些屏幕进行成像的系统。使用了两个部分的机器人来插入样品：SCARA 机器人用于将样品加载到显微镜载物台上，笛卡尔机器人用于将载物台放入电子显微镜中。使用了标准的 JEOL 1230 电子显微镜，尽管为载物台设计了新的尖端，并且重新连接了控制气闸的拨动开关以允许机器人控制。用于控制机器人的计算机程序与 Leginon 程序集成在一起，后者提供了一个用于自动对单个样品进行成像的模块。生成的图像被上传到 Sesame 实验室信息管理系统数据库中，与与结晶筛选相关的其他数据相关联。
