1. Deconstruction of Drop Volume Ratio/Temperature Optimization Experiments Joseph R. Luft, Edward H. Snell, Jennifer R. Wolfley, Meriem I. Said, Ann M. Wojtaszcayk, Raymond M. Nagel, Angela M. Lauricella, Steven A. Potter, Max H. Thayer, Christina K. Veatch, Michael G. Malkowski, and George T. DeTitta The Hauptman Woodward Medical Research Institute

2. Where are the losses? Screening Purified targets crystallized –36.6% ( 9234/25263) Optimization, Production Diffraction-quality crystals –18.9% (4780/25263) Diffraction –15.8% (3998/25263) Crystal Structure –14.5% (3665/25263) http://targetdb.pdb.org/statistics/TargetStatistics.html (Sept 7 th 2007) 100% 14.5%

3. Screening for crystallization Combine protein with cocktails Maximize chemical diversity Minimize time and protein J. Struct. Biol. (2003) 142: 170-179. Chayen, N. E., Shaw Stewart, P. D. & Blow, D. M. (1992). J. Cryst. Growth, 122, 176-180.

4. Screening cocktails A total of 1536 solutions Group I (233) –35 salts (3 conc); 8 pH’s Group II (737) –5 PEGs (2 conc); –36 salts (0.1M); 8 pH’s Group III (566) –Hampton Research Screens

5. Distribution of hits from 167 proteins # hits from 1536 screen # protein samples Optimization Goal: Leave no hit behind

6. High-throughput Optimization Screening lab output: March 2006-2007 –2118 proteins x 1536 cocktails –3.2 million screening experiments Keep pace with screening –Proteins often have several crystallization hits –Different cocktails produce different crystals Multi-parametric optimization to rapidly and systematically fine-screen conditions in chemical space that directly surrounds the screening hit

7. DVR/T Optimization Drop Volume Ratio / Temperature Protein Science (2007) 16: 715-722. Rayment, I, Structure, (2002) 10 (2): 147-151.

8. 96 proteins at a time 80 fine-screened conditions x 5 plates Incubated at: 4,14, 23, 30, 37 o C

9. V P > V C V C > V P Representing the data for 1 of 96 Cocktail = 100mM HEPES, pH 7.5, 100 mM Mg 2 Cl 2, 20% (w/v) PEG 8000 Protein = 20 mg/mL P6891 in 20mM Tris-HCl, pH 7.6, 0.02% NaAzide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4 o C 14 o C 23 o C 30 o C 37 o C

10. Protein + Cocktail assigns normal or retro-solubility Cocktail A = 100mM Na Acetate, pH 5.0, 100 mM NH 4 SCN, 20% (w/v) PEG 4000 Cocktail B = 100mM MOPS, pH 7.0, 100mM NH 4 Br, 80% (v/v) PEG 400 Protein = 20mg/mL P6306 in 10mM Tris, pH 7.4, 100mM NaCl

11. What is the success rate? 44 proteins that produced hits from HTS For 13 of the 44 proteins, crystals were visually improved using DVR/T Phase information is generated even in cases where the crystal quality doesn’t improve Phase information directs the route for subsequent optimization efforts

12. Protein = 13.5 mg/mL P6892 in 20mM Tris-HCl, pH 7.6, 300mM NaCl Cocktail = 100mM MES, pH 6.0, 100 mM Magnesium Acetate, 20% (w/v) PEG 8000 HTS ratio 8, 23 o CDVR/T ratio 8, 4 o C Ratio constant and temperature changes

13. Protein = 20 mg/mL P6512 in 10mM Tris-HCl, pH 7.5, 0.02% Na Azide Cocktail = 100mM HEPES, pH 7.5, 100 mM Rubidium chloride, 40% (w/v) PEG 20,000 HTS ratio 8, 23 o CDVR/T ratio 5, 23 o C Ratio changes and temperature constant

14. HTS ratio 8, 23 o CDVR/T ratio 4, 14 o C Protein = 13.5 mg/mL P6892 in 20mM Tris-HCl, pH 7.6, 300mM NaCl Cocktail = 100mM Tris-HCl, pH 8.0, 100 mM Magnesium Nitrate, 40% (w/v) PEG 20000 Ratio changes and temperature changes

15. Protein = 20.3 mg/mL P6891 in 20mM Tris-HCl, pH 7.6, 100mM NaCl Cocktail = 100mM HEPES, pH 7.5, 100 mM Magnesium chloride, 20% (w/v) PEG 8000 HTS ratio 8, 23 o CDVR/T ratio 3, 14 o C Ratio changes and temperature changes

16. How does DVR/T relate to a phase diagram?

17. Phase data relevant to crystallization 80 experiments centered on the screening hit 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4 o C 14 o C 23 o C 30 o C 37 o C Cocktail = 100mM CAPS, pH 10.0, 100 mM Ammonium phosphate (dibasic), 20% (w/v) PEG 20000 Protein = 20mg/mL P6512 in 10mM Tris-HCl, pH 7.5, 0.02% Na Azide = screening hit

18. Undersaturated or Metastable 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4 o C 14 o C 23 o C 30 o C 37 o C Cocktail = 100mM CAPS, pH 10.0, 100 mM Ammonium phosphate (dibasic), 20% (w/v) PEG 20000 Protein = 20mg/mL P6512 in 10mM Tris-HCl, pH 7.5, 0.02% Na Azide

19. Labile Zone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4 o C 14 o C 23 o C 30 o C 37 o C Cocktail = 100mM CAPS, pH 10.0, 100 mM Ammonium phosphate (dibasic), 20% (w/v) PEG 20000 Protein = 20mg/mL P6512 in 10mM Tris-HCl, pH 7.5, 0.02% Na Azide

20. Precipitation Zone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4 o C 14 o C 23 o C 30 o C 37 o C Cocktail = 100mM CAPS, pH 10.0, 100 mM Ammonium phosphate (dibasic), 20% (w/v) PEG 20000 Protein = 20mg/mL P6512 in 10mM Tris-HCl, pH 7.5, 0.02% Na Azide

21. Empirical solubility data for seeding streak seed from ratio 7 to ratio 4 time = 0 time = 17 hrs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Protein = 10.2 mg/mL P6893 in 20mM Tris-HCl, pH 7.6 Cocktail = 100mM MOPS, pH 7.0, 100 mM Lithium sulfate, 40% (w/v) PEG 4000

22. What are the DVR/T variables? Any solute protein = solute cocktail = variable –[ Protein ] –[ Precipitating agent ] –[ Buffers ] –[ Chemical additives ] A series of experiments with: 1) Temperature constant Chemistry variable 2) Temperature variable Chemistry constant Temperature pH

23. Solutes’ concentrations (starting) [P] init 10.0 mg/mL [C] init 1.00 M

24. Dehydration of the experiment drops Batch, but dehydration still occurs Rate is temperature-dependent Solutes’ concentrations steadily increase time [solutes] time

25. Day 1 Cocktail = 100mM Tris, pH 8.0, 100 mM Na 2 S 2 O 3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide 4 o C 14 o C 23 o C 30 o C 37 o C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

26. Day 7 Cocktail = 100mM Tris, pH 8.0, 100 mM Na 2 S 2 O 3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide 4 o C 14 o C 23 o C 30 o C 37 o C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

27. Day 14 Cocktail = 100mM Tris, pH 8.0, 100 mM Na 2 S 2 O 3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide 4 o C 14 o C 23 o C 30 o C 37 o C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

28. Day 21 Cocktail = 100mM Tris, pH 8.0, 100 mM Na 2 S 2 O 3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide 4 o C 14 o C 23 o C 30 o C 37 o C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

29. Day 28 Cocktail = 100mM Tris, pH 8.0, 100 mM Na 2 S 2 O 3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide 4 o C 14 o C 23 o C 30 o C 37 o C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

30. Day 60 Cocktail = 100mM Tris, pH 8.0, 100 mM Na 2 S 2 O 3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide 4 o C 14 o C 23 o C 30 o C 37 o C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

31. DVR/T inherently fine-screens pH (effect of buffer concentration and temperature) 4 o C 37 o C V P > V C V C > V P

32. Small differences in cocktail pH Big differences in outcomes Cocktails: 100mM NH 4 H 2 PO 4, 100mM CAPS, pH= _____, 20%(v/v) PEG 20k Protein: 20mg/ml P6512, 10mM Tris, pH 7.5, 0.02% Sodium Azide pH 9.80 10.00 10.25 10.50 9.80 10.00 10.25 10.50 pH 23 o C

33. Conclusions Use same crystallization method / solutions for both screening and optimization Simple/rapid set up Optimization is centered on screening hit –Screening hit expanded to 80 conditions without additional formulation Variables may be difficult to quantify, but can be precisely, volumetrically reproduced Guide for second-tier optimization

34. Acknowledgements Work supported in part by: NIH GM074899, John R. Oishei Foundation, Margaret L. Wendt Foundation, and the James H. Cummings Foundation. Special thanks to the following people for their enthusiastic and valuable collaborations: –Bob Cudney (Hampton Research) –Rachel Cochran (Matrix Technologies) –Brian Wright (Brook-Anco) –Ulrike Honisch, Guenther Knebel, and Bob Brino (Greiner-BioOne)