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Experimental mapping of protein precipitation diagrams
Morten O.A. Sommer ([email protected]) Centre for Crystallographic Studies University Of Copenhagen
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Look at protein crystallography and liquid handling
Low volume liquid handling technology more experiments performed using less SAMPLE
Lab automation more experiments performed using less TIME
Low TIME and SAMPLE consumption enables new approaches to protein crystallization
Liquid handling for protein crystallization
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Microfluidic formulator technology
1 mm
Experiments done by: Carl Hansen, Morten Sommer and Stephen Quake. PNAS (2004) 101:14431-14436
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Experiments done by: Carl Hansen, Morten Sommer and Stephen Quake. PNAS (2004) 101:14431-14436
Metering accurate and robust
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Injection volume: 80 pL +/- 0.6 pL
Metering accuracy determined by absorption measurements
Water at 20 degrees C
Motor oil (SAE 20) at 20 degrees C
Raw Linseed oil 20 degrees C
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Ideal approach to protein crystallization
• GOAL: Further rationalization of protein crystallization
• Using minute amounts of protein sample to quantify:– Protein stability, folding & activity– Protein physical chemistry (solubility and
precipitation limits) – Protein - protein interactions (Virial
coefficients etc.)
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Phase diagram of: aspartyl-tRNA synthetase-1
From Thermus thermophilus
Zhu et. al. 2001 Acta Cryst. D 57:552-558
Why use precipitation diagrams?
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Detecting precipitation
Detection of Precipitation
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Titration NumberS
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Towards a rational approach: Tailor made screens based on
precipitation diagrams
• Characterize protein solution and identify potential conditions
• Map protein precipitation diagrams
• Design and set up a tailor made crystallization screen based on the precipitation diagrams of the particular protein
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Experiments done by: Carl Hansen, Morten Sommer and Stephen Quake. PNAS (2004) 101:14431-14436
Initial validation: Xylanase
1. Make solubility fingerprint identifying precipitating chemical conditions
2. Map precipitation diagrams for potential conditions
3. Set up crystallization experiments near precipitation boundary
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Initial validation: XylanaseCrystallization probability pr. trial
OPT (Tailor made screen): 27 hits out of 48 experiments = 56 %
Sparse matrix screens: 3 hits out of 384 experiments = 0.8 %
Experiments done by: Carl Hansen, Morten Sommer and Stephen Quake. PNAS (2004) 101:14431-14436
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Further validation Membrane protein: SERCA
• Study the crystallization of membrane proteins using the previously crystallized calcium pump (SERCA)
• Crystallization conditions are know
• Reliable preparation and purification
Sørensen et.al., (2004) Science 304, 1672-1675
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Further validation Membrane protein: SERCA
• Solubility fingerprint can be used to identify specific protein – precipitant interactions
• Identification of specific interaction between sodium acetate and SERCA
• Sodium acetate is an established crystallization agent for SERCA
Experiments done by: Morten Sommer and Sine Larsen. Journ. of Synchrotron Rad. (2005) in press
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Further validation Membrane protein: SERCA
• Based on the characterization of specific protein – precipitant interactions several chemical conditions were selected for precipitation diagram mapping
• Set up tailor made crystallization screen• Identification of well known and new
crystallization agents• Potentially useful for crystallizing previously
uncrystallized membrane proteins
Experiments done by: Morten Sommer and Sine Larsen. Journ. of Synchrotron Rad. (2005) in press
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Process diagram
Proteinsample
Formulatorchip
Solubility fingerprint
Analysis ofprotein-precipitant
interaction
Precipitationdiagrams
Design rational crystallizationexperiments
Setupcrystallizationexperiments
Monitorexperiments
Crystals
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PerspectivesRational approach to protein crystallization using minute sample volumes
Rational approaches are possible for many targets that are available in low amounts (Membrane proteins, protein complexes, and proteins purified from native tissue).
TOTAL 35
Task Volume consumption (μL)
Solubility characterization 10
Setup of 300 crystallization exp. 25
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Testing previously uncrystallized membrane proteins
The ultimate test of the rational approach: 3 previously uncrystallized membrane proteins are tested.
1. Voltage gated channel 2. DsbB: disulfide bond forming
membrane protein. 3. AIDA: adhesin autotransporter
protein
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Voltage-gated channel:Solubility mapping
Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Jose Santos and Mauricio Montal
Voltage-gated channel in 0.1 M Linear Buffer pH 6.5
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Based on the solubility fingerprint 40 precipitation diagrams are mapped out.
Volume consumption pr. precipitation diagram: 100 nL
Total consumption for solubility screen and precipitation diagrams: 8 μL (44.8 μg)
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Voltage-gated channel:Crystallization experiments
Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Jose Santos and Mauricio Montal
Voltage-gated channel in 0.1 M Linear Buffer pH 6.5
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A tailor made screen of 288 conditions is designed. The screen is set up as sitting drop exp. using an ORYX 6 at Douglas Instruments using 17 μL sample (95 μg of protein)
An additional screen is set up testing different additives
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Voltage-gated channel:Crystallization experiments
Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Jose Santos and Mauricio Montal
Crystals tested at ESRF beamline ID 29.
Not protein crystals
Scalebars = 100 microns
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DsbB:Solubility mapping
DsbB in 0.1 M Linear Buffer pH 9 and 80 mM Calcium Acetate
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Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
DsbB in 0.1 M Linear Buffer pH 9 and 80 mM Calcium Acetate
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40 chemical conditions are chosen for determination of their precipitation diagram.
Using a total of 4 μL (40 μg of protein).
A tailor made screen consisting of 288 conditions was designed and set up using 18 uL (180 μg)
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DsbB:Crystallization experiments
Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
Crystals tested at ESRF ID 29
Some were not protein.
Some did not diffract cryo optimization
Scalebars = 100 microns
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AIDA:Solubility characterization
AIDA with 0.1 M Linear Buffer pH 4 and 8 % v/v Glycerol
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Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
40 precipitation diagrams are selected for mapping based on solubility fingerprint
Based on the diagrams a 576 experiment screen is designed and set up
Volume consumption:
Solubility mapping: 8 μL
Crystallization exp.: 22 μL
AIDA with 0.1 M Linear Buffer pH 4 and 8 % v/v Glycerol
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AIDA:Crystallization experiments
Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
Crystals tested at ESRF ID 29
Some did not diffract optimize cryo conditions
Scalebars = 100 microns
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Protein consumption
VGC DsbB AIDA
Solubility char.
>5000 exp. 8 μL (45μg)
>5000 exp. 8 μL (80μg)
>5000 exp. 8 μL (80μg)
Cryst. Exp ~576 exp.
34 μL(190μg)
~ 288 exp.
18 μL(180μg)
~ 576 exp.
22 μL(220μg)
Crystal Hits No Yes Yes
Total protein consumption
235 μg 260 μg 300 μg
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Summarizing remarks
• As liquid handling technologies have achieved ~1 nL experimental volumes. A rationalization of protein crystallization in terms of
precipitation diagrams is possible
• Rational approaches to protein crystallization are performed using < 300 μg of protein sample.
• Hope: This method and technology will allow for a better understanding of the crystallization process - and that complementary low volume technology will be developed to address other aspects of protein crystallization
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Acknowledgements• Univ. Of Copenhagen
– Jens-Christian Poulsen – Prof. Sine Larsen– Flemming Hansen– Centre for Crystallographic
Studies• Univ. Of Aalborg
– Prof. Daniel Otzen– Brian Vad
• Univ. Of Aarhus– Ass. Prof. Poul Nissen– Prof. Jesper Vuust Møller
• Tech. Univ. Of Denmark– Ass. Prof. Jörg Kutter– Detlef Snakenborg
• Stanford– Prof. Stephen R. Quake
• Univ. Of British Columbia– Ass. Prof. Carl L. Hansen
• Univ. of California – San Diego– Prof. Mauricio Montal– Dr. Jose Santos
• Douglas Instruments– James Smith – Peter Baldock – Patrick Shaw Stewart
• ESRF – ID29– Gordon Leonard
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Experimental mapping of protein precipitation diagrams
Morten O.A. Sommer ([email protected])Centre for Crystallographic Studies Univ. Of Copenhagen