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Deconstruction of Drop Volume Ratio/Temperature Optimization Experiments

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,

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Deconstruction of Drop Volume Ratio/Temperature Optimization Experiments

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  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) 100% 14.5% http://targetdb.pdb.org/statistics/TargetStatistics.html (Sept 7th 2007)

  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 Optimization Goal: Leave no hit behind # protein samples # hits from 1536 screen

  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 OptimizationDrop 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 Incubated at: 4,14, 23, 30, 37oC x 5 plates

  9. 4 oC 14 oC 23 oC 30 oC 37 oC Representing the data for 1 of 96 VP > VC VC > VP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Cocktail= 100mM HEPES, pH 7.5, 100 mM Mg2Cl2, 20% (w/v) PEG 8000 Protein = 20 mg/mL P6891 in 20mM Tris-HCl, pH 7.6, 0.02% NaAzide

  10. Protein + Cocktail assigns normal or retro-solubility Cocktail A = 100mM Na Acetate, pH 5.0, 100 mM NH4SCN, 20% (w/v) PEG 4000 Cocktail B = 100mM MOPS, pH 7.0, 100mM NH4Br, 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 oC DVR/T ratio 8, 4 oC 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 oC DVR/T ratio 5, 23 oC Ratio changes and temperature constant

  14. HTS ratio 8, 23 oC DVR/T ratio 4, 14 oC 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 oC DVR/T ratio 3, 14 oC Ratio changes and temperature changes

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

  17. 4 oC 14 oC 23 oC 30 oC 37 oC = screening hit Phase data relevant to crystallization80 experiments centered on the screening hit 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 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

  18. 4 oC 14 oC 23 oC 30 oC 37 oC Undersaturated or Metastable 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 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. 4 oC 14 oC 23 oC 30 oC 37 oC Labile Zone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 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. 4 oC 14 oC 23 oC 30 oC 37 oC Precipitation Zone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 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 seedingstreak seed from ratio 7 to ratio 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 time = 0 time = 17 hrs 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 soluteprotein = solutecocktail = variable • [ Protein ] • [ Precipitating agent ] • [ Buffers ] • [ Chemical additives ] • Temperature • pH A series of experiments with: 1) Temperatureconstant Chemistryvariable 2) Temperaturevariable Chemistryconstant

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

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

  25. 4 oC 14 oC 23 oC 30 oC 37 oC Day 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Cocktail= 100mM Tris, pH 8.0, 100 mM Na2S2O3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide

  26. Day 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4 oC 14 oC 23 oC 30 oC 37 oC Cocktail= 100mM Tris, pH 8.0, 100 mM Na2S2O3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide

  27. Day 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4 oC 14 oC 23 oC 30 oC 37 oC Cocktail= 100mM Tris, pH 8.0, 100 mM Na2S2O3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide

  28. Day 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4 oC 14 oC 23 oC 30 oC 37 oC Cocktail= 100mM Tris, pH 8.0, 100 mM Na2S2O3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide

  29. Day 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4 oC 14 oC 23 oC 30 oC 37 oC Cocktail= 100mM Tris, pH 8.0, 100 mM Na2S2O3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide

  30. Day 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4 oC 14 oC 23 oC 30 oC 37 oC Cocktail= 100mM Tris, pH 8.0, 100 mM Na2S2O3, 20% (w/v) PEG 4000 Protein = 60mg/mL P6513 in 10mM Tris-HCl, pH 7.5, 0.02% NaAzide

  31. 4 oC VP > VC VC > VP 37 oC DVR/T inherently fine-screens pH(effect of buffer concentration and temperature)

  32. Small differences in cocktail pHBig differences in outcomes pH 9.80 10.00 10.25 10.50 9.80 10.00 10.25 10.50 pH 23oC Cocktails: 100mM NH4H2PO4, 100mM CAPS, pH= _____, 20%(v/v) PEG 20k Protein: 20mg/ml P6512, 10mM Tris, pH 7.5, 0.02% Sodium Azide

  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)

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