title: An automated pipeline to screen membrane protein 2D crystallization.
abstract: 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.
authors.affiliation: The New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027, USA.
authors.forename: Changki
authors.initials: C
authors.lastname: Kim
authors.forename: Martin
authors.initials: M
authors.lastname: Vink
authors.forename: Minghui
authors.initials: M
authors.lastname: Hu
authors.forename: James
authors.initials: J
authors.lastname: Love
authors.forename: David L
authors.initials: DL
authors.lastname: Stokes
authors.forename: Iban
authors.initials: I
authors.lastname: Ubarretxena-Belandia
mesh_terms: D001331:Automation; D020285:Cryoelectron Microscopy; D003460:Crystallization; D018360:Crystallography, X-Ray; D008565:Membrane Proteins / Q000737:chemistry*
chemical_list: D008565:Membrane Proteins