W0189

Experiences With Commercial Robots for High Throughput Crystallization Effort for Structural Genomics. Ashit Shah, Florian Schubot, Shu-Huey Chang, Doowon Lee, Wolfram Tempel, Zhi-Jie Liu, John P. Rose and Bi-Cheng Wang, Southeast Collaboratory for Structural Genomics, Dept. of Biochemistry and Molecular Biology, Univ. of Georgia, Athens, GA 30602.

In the past year, we have tested 106 Pyrococcus furiosus and 6 human proteins in microbatch mode with Douglas ORYX1-6 crystallization robots using bar coded 72-well HLA plates. 76% (81/106 samples) of the Pyrococcus and 50% (3/6 samples) of the human proteins have been successfully crystallized. 31% (25) of the Pyrococcus protein crystals and 67% (2) of the human protein crystals diffract better than 3Å resolution. Initial screening was carried out using 288 conditions selected from 6 commercial sparse matrix screens – Crystal Screen, Memfac, Crystal Screen Cryo and Peg Ion from Hampton Research, and Wizard I & II from Emerald Biostructures. Crystallization conditions were then optimized using in-house grid screens, additive screen and response surface methodology. Reductive methylation of lysine’s was carried out for 12 proteins. Three proteins which had failed to crystallize previously, produced crystals and new crystallization conditions were obtained for four other proteins when their lysine’s were methylated. The diffraction quality of Pfu-1147304 and Pfu-1208389 has been improved by using Linkers. Crystallization results were recorded into Emerald Biostructure’s Crystal Monitor database. To enhance our crystallization and imaging capabilities, a Genomics Solutions Honeybee SD crystallization robot (set up crystallization plates) and a Crystal Farm harvesting robotic (store crystallization plates and auto/manually image the droplets into a database) have been installed recently. Results and experiences of using these commercially available robotic crystallization systems will be reported.