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

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

5. SGPP Lmaj004655AAASeMet

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

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,

10. Co-Crystallants Used in This Study01 02 04 05 09 10 11 12 13

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 RR NIH P50 GM and NIH P50 GM
Special thanks to Greiner Bio-One