banner
home > research > ht crystallization screening
HIGH-THROUGHPUT CRYSTALLIZATION SCREENING

Beginning the crystallization pipeline with a large number of screening solutions in which the variables are concentrations of the macromolecule, detergent, chemical additives, pH and temperature increases the likelihood of identifying multiple, chemically distinct crystallization conditions.  Crystals produced from different chemical environments often have different physical properties.  This benefits downstream optimization, soaking, cryo-preservation and ultimately X-ray diffraction; it provides the laboratory with multiple paths to reach the desired product, a diffraction-quality crystal.

Purified, soluble proteins are processed through a mature, high-throughput screening facility (located at the Hauptman-Woodward Medical Research Institute) to identify crystallization conditions.  Syringe-based liquid handling systems are used to prepare microbatch-under-oil crystallization experiments in 1536-well microassay plates.  Each experiment plate holds a single macromolecule combined with 1536 chemical solutions (cocktails).  Individual experiments are composed of 200 nanoliters of protein solution and 200 nanoliters of cocktail solution.  A screen of 1536 experiments is set up with 400 mL of protein solution (10 mg/mL).  The high-throughput laboratory currently screens 200 different macromolecules each month.  During the past 8 years, 15 million crystallization experiments were set up for 9700 macromolecules.

After setting up screening experiments, the outcomes are digitally recorded 1 day after the addition of protein and weekly thereafter for a period of 4 weeks using 3 custom-designed imaging systems. Each has the capacity to hold 28 plates and image at a rate of 2 plates (3,000 experiments) per hour.  Images are stored on multiple hard drives and archived on offline media.  Outcomes are reviewed to identify combinations of proteins and cocktails that demonstrate a propensity to crystallize.  Successful combinations advance through the structure-determination pipeline to the crystal-growth optimization stage.  Samples that fail to produce any screening outcomes suitable for optimization are re-cycled through protein expression and purification salvage pathways before re-entering the crystallization pipeline.