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Detergent concentration measurements
The detergent concentration is critical for optimizing the 2D crystallization conditions. It can be measured to satisfactory accuracy using the DropBox, a device that determines the contact angle of a drop deposited on Parafilm as described in Kaufmann et al. (2006) A novel method for detergent concentration determination. Biophys. J. 90(310):317.

Detergent Concentration affect on drop shape (Principles)

Detergent Concentration Measurement Systems(Mechanical Drawings)

Drop Shape Analysis for Detergent Concentration (MatLab Protocol)

Xtrace Drop Program (Python Protocol)

Dialysis Block
2D crystallization by dialysis can be achieved by different type of devices. TEMIMPS operates temperature controlled 96 and 30 well systems with typically 50 μl reaction mixture/well. More information about the dialysis block can be found in Vink et al.(2007) A high-throughput strategy to screen 2D crystallization trials of membrane proteins. J Struct Biol. 160(3):295-304.

Cyclodextrin driven 2D crystallization
Cyclodextrin neutralizes detergent molecules with accurate detergent-dependent stoichiometry. Therefore, the kinetics of the reconstitution processes is precisely controlled by quantitative addition of a cyclodextrin solution to the reaction mixture containing solubilized proteins and lipids. The method requires only 5 μl/well. Two robots handling 96- or 384-well plates will be installed within TEMIMPS. More information can be found in: Iacovache et al.(2010) The 2DX robot: A membrane protein 2D crystallization Swiss Army knife. Journal of Structural Biology 169(370):378.

Automated Staining Robots
Members of the TEMIMPS consortium have developed staining robots preparing up to 96 grids. The heart of the staining robot shown is a dispenser combined with an aspirating device that allows grids to be extensively washed before staining. Magnets keep the grids in a holder for sample deposition, washing and staining. The robot is mounted on a Nano25 gantry robot from 157 Hudson Control Group (NJ, USA). More information cna be found in : Kim et al.(2010) An automated pipeline to screen membrane protein 2D crystallization. J Struct Funct Genomics. 11(2):155-66. and Coudray et al. (2010) Automated screening of 2D crystallization trials using transmission electron microscopy: A high-throughput tool-chain for sample preparation and microscopic analysis. J Struct Biol. 173:365-74.

John Henry
John Henry is a sample screening robot. It consists of three parts. A SCARA robot uses a vacuum probe to pick up individual EM grids from a 96-well tray and secure them in a customized EM holder. A Cartesian robot picks up the holder and manipulates it through the air lock and into the column of the electron microscope. A computer program developed at the Scripps Research Institute then selects suitable areas on the EM grid, focuses the electron microscope, and records a series of images using the CCD camera. It takes 30-45 min to record ~50 images from each grid. The images end up in a database managed by our Laboratory Information Management System (LIMS). This system associates the images not only with the particular conditions used for crystallization, but also with information about the protein construct and the protocols that were used for expression and purification. In the near future, we hope to develop computer vision algorithms that will evaluate the micrographs and assign scores that reflect the results of each crystallization trial. More information can be gfound in: Hu et al.(2010) Automated electron microscopy for evaluating two-dimensional crystallization of membrane proteins. J Struct Biol. 171(1):102-110.

'2D Crystallization of Membrane Proteins' Database

Data mining from a recent review by Abeyrathne et al., which tabulated successful 2D crystallization experiments from the literature, allowed us to create a database containing fields to express the physical and chemical properties of the key parameters in a 2D crystallization experiment. These include lipid composition, detergent, protein type, buffer composition (pH, salts, additives), temperature, and LPR (lipid-to-protein ratio). We also included the outcome of the experiments (crystal symmetry and unit cell dimensions), and properties of the ternary mixture between the protein (molecular weight, number of amino acids in transmembrane region, pI, etc), the lipids (alkyl chain length and saturation, headgroup, etc), and the detergent (CMC, aggregation number, etc). The database combines more than 200 crystallization conditions from about 200 papers and 150 membrane protein sequences.

For a quick start help, check our twikipage, or, for more information, download NYSBC_2d-crystallization_database.pdf.