title: Automated electron microscopy for evaluating two-dimensional crystallization of membrane proteins.
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.
authors.affiliation: New York Structural Biology Center, New York, NY 10027, USA.
authors.forename: Minghui
authors.initials: M
authors.lastname: Hu
authors.forename: Martin
authors.initials: M
authors.lastname: Vink
authors.forename: Changki
authors.initials: C
authors.lastname: Kim
authors.forename: Kd
authors.initials: K
authors.lastname: Derr
authors.forename: John
authors.initials: J
authors.lastname: Koss
authors.forename: Kevin
authors.initials: K
authors.lastname: D'Amico
authors.forename: Anchi
authors.initials: A
authors.lastname: Cheng
authors.forename: James
authors.initials: J
authors.lastname: Pulokas
authors.forename: Iban
authors.initials: I
authors.lastname: Ubarretxena-Belandia
authors.forename: David
authors.initials: D
authors.lastname: Stokes
mesh_terms: D001331:Automation; D003460:Crystallization; D008565:Membrane Proteins / Q000737:chemistry*; D008854:Microscopy, Electron / Q000295:instrumentation / Q000379:methods*; D017434:Protein Structure, Tertiary; D012984:Software
chemical_list: D008565:Membrane Proteins