Automated electron microscopy for evaluating two-dimensional crystallization of membrane proteins

New York Structural Biology Center, New York, NY 10027, USA.
Journal of Structural Biology (Impact Factor: 3.23). 07/2010; 171(1):102-10. DOI: 10.1016/j.jsb.2010.02.018
Source: PubMed

ABSTRACT 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.

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Available from: Martin Vink, Sep 26, 2015
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    • "After the detergent concentration has dropped below the CMC, screen for the formation of 2D crystals by electron microscopy of negatively stained preparations. This can also be done using a robotic setup (Hu et al., 2010; Iacovache et al., 2010). "
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    ABSTRACT: Electron crystallography is a powerful technique for the structure determination of membrane proteins as well as soluble proteins. Sample preparation for 2D membrane protein crystals is a crucial step, as proteins have to be prepared for electron microscopy at close to native conditions. In this review, we discuss the factors of sample preparation that are key to elucidating the atomic structure of membrane proteins using electron crystallography.
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    • "grid tray and load them into the specimen holder aided by an actuator to raise and lower the specimen holder lever arm. A second implementation, developed at the New York Structural Biology Center (NYSBC) shares the same principle but uses a Cartesian robot (which moves back and forth along a single, fixed axis) for the task of inserting the loaded grid holder and a Selective Compliant Assembly Robot Arm (SCARA, limited to motion within a defined xy-plane) for loading grids from the grid tray to the holder (Hu et al., 2010). Some advantages of subdividing the grid loading and holder insertion tasks include a decrease in the requirements for robotic alignments, an increase in fine-tuned motion specific to EM grid loading, and an overall decrease in cost. "
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