Transcript Document

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
Where are the losses?
100%
14.5%
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)
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
# protein samples
Optimization Goal: Leave no hit behind
# hits from 1536 screen
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 Optimization
Drop 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
Incubated at:
4,14, 23, 30, 37oC
x 5 plates
Representing the data for 1 of 96
VP > VC
1
2
3
VC > VP
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 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
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
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
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
HTS ratio 8, 23 oC
DVR/T ratio 4, 14 oC
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 crystallization
80 experiments centered on the screening hit
= 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
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 seeding
streak 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
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
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]
• Solutes’ concentrations steadily increase
time
time
Day 1
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
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
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
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
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
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
DVR/T inherently fine-screens pH
(effect of buffer concentration and temperature)
4 oC
VP > VC
VC > VP
37 oC
Small differences in cocktail pH
Big differences in outcomes
pH
pH
9.80
9.80
10.00
10.25
10.50
10.00
10.25
10.50
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
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)