1. Origin of a Species: History and observations of one high throughput crystallization laboratory J. R. Luft, R. J. Collins, S. M. Gulde, A.M. Lauricella, C. A. Mancuso, J. L. Smith, C. K. Veatch, and G. T. DeTitta Recent Advances in Macromolecular Crystallization Le Bischenberg, France May 8-11, 2005

2. Hypothesis By executing a large number of precipitation reactions for a large number of proteins we could predict crystallization conditions for a previously un-crystallized protein by comparison of precipitation behavior. Protein Binary Code PSS Unknown 1011010010110010-- Protein B 011101011101001011 Protein A 0001100101010101 5

3. What is the most efficient way to execute precipitation reactions? Batch, in particular microbatch in a capillary –Minimize solution volume requirements for precipitation cocktails

4. Good, but not Great Maximum throughput (manual) 50 experiments/technician/day Crystals were a ‘by-product’.

5. Microbatch Under Oil Chayen, N.E., Stewart, P.D.S., Blow, D.M., Journal of Crystal Growth, 122 (1992) 176-180. Minimal solution volumes Plates for storage and handling

6. Mothers and Daughters Mother (source) / Daughter (destination) plates Efficient parallel setups … source destination

7. Solution Delivery Pump Slot pins Pipettes Syringes Faster Syringes http://www.vp-scientific.com/index.htm

8. Screening for Crystallization Leads * 1536 1.Oil 2.Crystallization Cocktail 3.Macromolecular Solution

9. Quantity How Many Experiments? –20 plates per day (~ 200/month) –200(plates) x 1536(experiments) x 6(images) Generates 1.8 million images/month As of 3/17/2005: 4877 (plates) x 1536 (experiments) 7.5 million experiments 45 million images 469 active users

10. Setups per Month

11. HT Infrastructure at HWI

12. Variables 1.Plate 2.[Cocktail] 3.Oil 4.Sample [salt] 5.Sample State (solid or liquid) [M] [C]

13. 1. The Plate February 2004 –Square to circular cross-section well Better visibility Now in production at Greiner BioOne –LBR plates “significantly higher water absorption” 1 2

14. Image Quality 2000 2005 1.8 mm 0.7 mm

15. LBR Plate Imaged with Cross-Polarized Light

17. 2. Cocktail Dehydration Plates prepared with oil Cocktail added to plates (200nl) Plates are stored at 4 o C for 1 day – 2 weeks [M] [C] Dehydrate C drop Decrease V c drop Increase [C] Decrease dilution of [M] Increase [M] exp ?

18. 3. The Oil Paraffin Oil –(IR Spec grad) Emulsion formed with some batches Mineral Oil –(USP grade) December 2004

19. 4. [Salt] in the Samples Compared salt concentrations for 392 samples –([KCl] + [NaCl]) mM All samples were set up using the ‘standard’ 1536 screen and outcomes reviewed

21. Compared 158 samples shipped: –76 Frozen (dry ice) –82 Liquid (wet ice) All samples were set up using the ‘standard’ 1536 screen and outcomes reviewed 5. State of the Samples

22. Frozen Samples versus Liquid Samples frozen liquid Samples with hits Samples with no hits Number of Samples

23. Distribution # of Hits 45% had 8 or fewer leads from 1536 experiments

24. In Conclusion…

25. Work supported in part by the John R. Oishei Foundation, the Cummings Foundation NIH RR016924, NIH P50 GM-62413 and NIH P50 GM-64655 Special thanks to Bob Cudney Greiner Bio-One And …. Acknowledgements http://www.hwi.buffalo.edu