title: A high-throughput strategy to screen 2D crystallization trials of membrane proteins.
abstract: Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.
authors.affiliation: The New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Vink
authors.forename: Kd
authors.initials: K
authors.lastname: Derr
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: D019611:Archaeoglobus fulgidus / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000648:ultrastructure; D003460:Crystallization / Q000295:instrumentation / Q000379:methods*; D003461:Crystallography / Q000379:methods*; D003902:Detergents; D003956:Dialysis; D004867:Equipment Design; D007091:Image Processing, Computer-Assisted; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D016624:Negative Staining; D012242:Rhodobacter sphaeroides / Q000737:chemistry; D012371:Robotics; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; C071263:CopA protein, Bacteria; D003902:Detergents; D045342:Light-Harvesting Protein Complexes; D008565:Membrane Proteins