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Beginning the crystallization step in the structure-determination 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 investigator with multiple paths to reach the desired product, a diffraction-quality crystal.
The High-Throughput Crystallization Screening (HTS) Laboratory, located at the Hauptman-Woodward Institute (HWI), is a well-established resource for the structural genomics and structural biology communities. The HTS Lab currently screens about 200 purified macromolecular samples each month using syringe-based liquid handling systems to prepare microbatch-under-oil crystallization experiments in 1536-well microassay plates (PMID: 12718929). 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). Since February 2000, more than 19 million crystallization experiments have been set up for more than 12,500 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. Investigators are automatically notified by e-mail when images are ready for download from the HTS Laboratory FTP server. Digital images are reviewed using software (MacroscopeJ) packaged with the images and developed at the HWI to identify crystallization leads.
Since the summer of 2009, the HTS Lab has offered a special 1536-well, detergent-based screen for membrane proteins in addition to the standard screen used for soluble proteins.
The HTS Laboratory served as a crystallization resource for the Structural Genomics of Pathogenic Protozoa (SGPP) project and the Northeast Structural Genomics consortium (NESG) in phase I of the PSI and also served as the hub of the Center for High-Throughput Structural Biology (CHTSB), one of six specialized centers in phase II of the PSI. In the PSI:Biology era, the HTS Laboratory continues to provide crystallization support for soluble targets of NESG as well as for transmembrane protein targets of the New York Consortium on Membrane Protein Structure (NYCOMPS) and the Center for the Structures of Membrane Proteins (CSMP). Crystallization screening is also available for the greater structural biology community.