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High Throughput Crystallization Laboratory: History and Observations

This paper discusses the origin and observations of a high throughput crystallization laboratory, including the execution of precipitation reactions for predicting crystallization conditions. It explores the most efficient way to execute precipitation reactions and the use of microbatch under oil for minimal solution volumes. The paper also covers solution delivery methods, screening for crystallization leads, and variables in the laboratory setup. It concludes with the analysis of plate design, cocktail dehydration, salt concentrations in samples, and the state of the samples.

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High Throughput Crystallization Laboratory: History and Observations

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  1. Origin of a Species: History and observations of one high throughput crystallization laboratoryJ. 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 0111010111010010 11 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 • Minimal solution volumes • Plates for storage and handling Chayen, N.E., Stewart, P.D.S., Blow, D.M., Journal of Crystal Growth, 122 (1992) 176-180.

  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 • Oil • Crystallization Cocktail • Macromolecular Solution * 1536

  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 • Plate • [Cocktail] • Oil • Sample [salt] • 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 1.8 mm 0.7 mm 2000 2005

  15. LBR Plate Imaged with Cross-Polarized Light

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

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

  18. 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

  19. 5. State of the Samples • 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

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

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

  22. In Conclusion…

  23. Acknowledgements • 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 …. http://www.hwi.buffalo.edu

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