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Use of High Throughput Screening to Determine Lead Crystallization Conditions and as a Tool for Basic Research

Use of High Throughput Screening to Determine Lead Crystallization Conditions and as a Tool for Basic Research. HT Infrastructure: The Wet Lab. Sample Database. BIG BROTHER. Reader Tables RAID1. Failover fileserver. 2 TB RAID5 snapshots. HT Infrastructure: Computer Hardware and Software.

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Use of High Throughput Screening to Determine Lead Crystallization Conditions and as a Tool for Basic Research

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  1. Use of High Throughput Screening to Determine Lead Crystallization Conditions and as a Tool for Basic Research

  2. HT Infrastructure:The Wet Lab

  3. Sample Database BIG BROTHER Reader Tables RAID1 Failover fileserver 2 TB RAID5 snapshots HT Infrastructure:Computer Hardware and Software BACKUP Tape Optical Disc Offsite Data to Primary fileserver 1 TB RAID5 subsystem

  4. New Old How can we improve our output? Improved image quality = Improved lead detection

  5. SGPP Lmaj004655AAASeMet

  6. Images courtesy of Greiner Bio-One PS Glass LBR Outcome Verification Identify the outcome as crystalline Identify the composition

  7. Samples Screened – All Users

  8. HTS: Success Rate

  9. Co-Crystallants • The SGPP co-crystallants were designed to promote 2 types of macromolecular interactions: • intra-molecular interactions (freezers) and • inter-molecular interactions (glues) • The library uses “privileged structural motifs”, fused-ring aromatics (triazoliums or guanidiniums) “decorated with bromines”*. *Hovey, B., Verlinde, C. L. M. J., Merritt, E. A. & Hol, W. G. J. (1999). Structure-based discovery of a pore-binding ligand: Towards assembly inhibitors for cholera and related AB5 toxins. J. Mol. Biol. 285, 1169-1178.

  10. 01 04 02 10 09 05 11 12 13 Co-Crystallants Used in This Study

  11. Co-Crystallant Experiments • Crystallization in HTS laboratory • Compared the crystallization behavior of: • native protein • protein-DMSO • protein-DMSO-10mM Co-Crystallant • DLS • Look for evidence of aggregation in the presence of the co-crystallants.

  12. DLS Results • Sample aggregation occurred in the presence of the co-crystallants.

  13. DLS Results and Crystallization Outcomes: Ldon1686AAA (22)

  14. Venn Diagram of Crystallization Results: Ldon1686AAA (22)

  15. Venn Diagram of Crystallization Results:Hen Egg White Lysozyme

  16. Acknowledgements • Robert J. Collins, Nancy A. Fehrman, Stacey M. Gulde, Angela M. Lauricella, Carrie A. Mancuso, Christina K. Veatch and George T. DeTitta • Jizhen Li, Brian Carroll, Erkang Fan, Mark Robien and Wim G.J. Hol

  17. Acknowledgements • Work supported in part by the John R. Oishei Foundation, the Cummings Foundation, the Western New York Foundation, NASA NAG8-1594, NASA NAG8-1839, NASA NCC8-232, NIH RR016924 NIH P50 GM-62413 and NIH P50 GM-64655. • Special thanks to Greiner Bio-One

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