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Overview

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Overview

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  1. What We’ve Learned from the First 717,312 Crystallization ExperimentsGeorge T. DeTitta1, Melissa A. Bianca1, Robert J. Collins1, Ann Marie E. Faust1, Jillian N. Kaczmarek1, Joseph R. Luft1, Nancy A. Urban1, Walter A. Pangborn1, Jennifer R. Wolfley1, Igor Jurisica2, Patrick Rogers2, Gerald Quon2, Janice Glasgow3, Suzanne Fortier3(1)   Hauptman-Woodward Medical Research Institute, 73 High Street, Buffalo, NY 14203-1196 USA (2)   Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, Division of Cancer Informatics, 610 University Avenue, Room 8-413 Toronto, Ontario M5G 2M9 Canada (3) Queen’s University, Kingston, Ontario K7L 3N6 Canada

  2. Overview The HTP Crystallization Laboratory at HWI

  3. Microbatch Under Oil Chayen, N.E., Stewart, P.D.S., Blow, D.M., Journal of Crystal Growth, 122 (1992) 176-180.

  4. The Experiment Plate Each plate contains one sample x 1536 unique chemical cocktails

  5. Setting Up the Plates • Source/Destination Plate Methods • Reformat Plates from Low to High Density • Modified Robbins Scientific Hydra 96 and 384 are used to deliver the solutions

  6. Hydra 384 Liquid Handling System

  7. Imaging the Plates

  8. MacroScope

  9. Enlarged Image with Details

  10. Animated Views • 1536 different Bovine liver catalase crystallization experiments were setup. • Every experiment was recorded 203 times at 30 minute intervals. • Still images were used to produce time-lapse recordings of the experiments. • Determine preliminary outcomes that could lead to crystallization.

  11. Improvements • Decreased Solution Volume • Cocktails • Image Quality • Image Viewing and Sorting • Image Analysis

  12. Underlying Physical Parameters • Activity of water • Viscosity • Surface Tension • pH • Ionic Strength • Hofmeister Series • Excluded Volume • Specific Ion Effects

  13. Ancillary Data Collection Techniques used to characterize proteins • SDS-PAGE • Dynamic Light Scattering Instruments used to characterize cocktails • Refractometer • Vapor Pressure Osmometer • Tensiometer • Viscometer

  14. Examples of Outcomes

  15. User Profile GroupLaboratory Use Structural biology 54.8% Structural genomics 38.5% Pharmaceutical 6.7%

  16. Conclusions • HTP crystallization can have dual roles: 1) A method to produce crystals 2) A tool for knowledge discovery • Setting up a lot of crystallization experiments is not a cure-all (GIGO). • Method variation can produce different results. • Image quality is critical for success. • Verification of the outcomes’ composition is an important focus for development.

  17. Acknowledgements • John R. Oishei Foundation • Cummings Foundation • NASA NAG8-1594 • NIH grant 1 P50 GM62413-01 • NIH grant 1 P50 GM64655-01 • IBM SUR • NSERC (Natural Sciences and Engineering Research Council of Canada) • CITO (Communications and Information Technology Ontario)

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