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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, - PowerPoint PPT Presentation
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Deconstruction of Drop Volume Ratio/Temperature Optimization
ExperimentsJoseph 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
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 7th 2007)
100% 14.5%
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.
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
Distribution of hits from 167 proteins
# hits from 1536 screen
# pr
otei
n sa
mpl
esOptimization Goal: Leave no hit behind
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
DVR/T OptimizationDrop Volume Ratio / Temperature
Protein Science (2007) 16: 715-722.
Rayment, I, Structure, (2002) 10 (2): 147-151.
96 proteins at a time 80 fine-screened conditions
x 5 plates
Incubated at:
4,14, 23, 30, 37oC
VP > VC VC > VP
Representing the data for 1 of 96
Cocktail = 100mM HEPES, pH 7.5, 100 mM Mg2Cl2, 20% (w/v) PEG 8000Protein = 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 oC
14 oC
23 oC
30 oC
37 oC
Protein + Cocktail assigns normal or retro-solubility
Cocktail A = 100mM Na Acetate, pH 5.0, 100 mM NH4SCN, 20% (w/v) PEG 4000Cocktail 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
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
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
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
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
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
How does DVR/T relate to a phase diagram?
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
4 oC
14 oC
23 oC
30 oC
37 oC
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
Undersaturated or Metastable
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 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
Labile Zone
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 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
Precipitation Zone
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 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
Empirical solubility data for seedingstreak 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
What are the DVR/T variables?
• Any soluteprotein = solutecocktail = variable
– [ Protein ]– [ Precipitating agent ]– [ Buffers ]– [ Chemical additives ]
A series of experiments with:
1) Temperatureconstant Chemistryvariable
2) Temperaturevariable Chemistryconstant
•Temperature•pH
Solutes’ concentrations (starting)
[P]init
10.0 mg/mL
[C]init 1.00 M
Dehydration of the experiment drops
• Batch, but dehydration still occurs
• Rate is temperature-dependent
• Solutes’ concentrations steadily increase
time
[sol
utes
]
time
Day 1
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
4 oC
14 oC
23 oC
30 oC
37 oC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Day 7
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
4 oC
14 oC
23 oC
30 oC
37 oC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Day 14
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
4 oC
14 oC
23 oC
30 oC
37 oC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Day 21
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
4 oC
14 oC
23 oC
30 oC
37 oC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Day 28
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
4 oC
14 oC
23 oC
30 oC
37 oC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Day 60
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
4 oC
14 oC
23 oC
30 oC
37 oC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
DVR/T inherently fine-screens pH(effect of buffer concentration and temperature)
4 oC
37 oC
VP > VC VC > VP
Small differences in cocktail pHBig differences in outcomes
Cocktails: 100mM NH4H2PO4, 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
9.8010.0010.2510.50
pH 23oC
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
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)
