Presentation is loading. Please wait.

Presentation is loading. Please wait.

Experimental mapping of protein precipitation diagrams Morten O.A. Sommer Centre for Crystallographic Studies University Of

Published byJamari Bolling Modified over 9 years ago

Similar presentations

Presentation on theme: "Experimental mapping of protein precipitation diagrams Morten O.A. Sommer Centre for Crystallographic Studies University Of"— Presentation transcript:

1 Experimental mapping of protein precipitation diagrams Morten O.A. Sommer (morten@ccs.ki.ku.dk) Centre for Crystallographic Studies University Of Copenhagenmorten@ccs.ki.ku.dk

2 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

3 Microfluidic formulator technology 1 mm Experiments done by: Carl Hansen, Morten Sommer and Stephen Quake. PNAS (2004) 101:14431-14436

4 Metering accurate and robust 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

5 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.)

6 Phase diagram of: aspartyl- tRNA synthetase-1 From Thermus thermophilus Zhu et. al. 2001 Acta Cryst. D 57:552-558 Why use precipitation diagrams?

7 Detecting precipitation

8 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

9 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

10 Initial validation: Xylanase Crystallization 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

11 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

12 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

13 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

14 Process diagram Protein sample Formulator chip Solubility fingerprint Analysis of protein-precipitant interaction Precipitation diagrams Design rational crystallization experiments Setup crystallization experiments Monitor experiments Crystals

15 Perspectives Rational 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). TOTAL35 TaskVolume consumption (μL) Solubility characterization10 Setup of 300 crystallization exp.25

16 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

17 Voltage-gated channel: Solubility mapping Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Jose Santos and Mauricio Montal 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)

18 Voltage-gated channel: Crystallization experiments Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Jose Santos and Mauricio Montal 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

19 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

20 DsbB: Solubility mapping Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Brian Vad and Daniel Otzen 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)

21 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

22 AIDA: Solubility characterization 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

23 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

24 Protein consumption VGCDsbBAIDA Solubility char. >5000 exp. 8 μL (45μg) >5000 exp. 8 μL (80μg) Cryst. Exp~576 exp. 34 μL(190μg) ~ 288 exp. 18 μL(180μg) ~ 576 exp. 22 μL(220μg) Crystal HitsNoYes Total protein consumption 235 μg260 μg300 μg

25 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

26 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

27 Experimental mapping of protein precipitation diagrams Morten O.A. Sommer (morten@ccs.ki.ku.dk) Centre for Crystallographic Studies Univ. Of Copenhagen

Download ppt "Experimental mapping of protein precipitation diagrams Morten O.A. Sommer Centre for Crystallographic Studies University Of"

Similar presentations

Gas Solubilities Henry’s Law: [A]equilibrium = SA · pA

Gas Solubilities Henry’s Law: [A]equilibrium = SA · pA
Douglas Instruments Microseeding slide 1 Microseed it! Introduction to microseeding Patrick Shaw Stewart Douglas Instruments Limited (near Oxford, UK):

Douglas Instruments Microseeding slide 1 Microseed it! Introduction to microseeding Patrick Shaw Stewart Douglas Instruments Limited (near Oxford, UK):
Douglas Instruments Microbatch seminar- slide 1 Introduction to microbatch protein crystallization Patrick Shaw Stewart Imperial College, London: Professor.

Douglas Instruments Microbatch seminar- slide 1 Introduction to microbatch protein crystallization Patrick Shaw Stewart Imperial College, London: Professor.
Microfluidic Dialysis Protein Crystallization Jiang Huang GN Biosystems, Inc. March 26, 2009.

Microfluidic Dialysis Protein Crystallization Jiang Huang GN Biosystems, Inc. March 26, 2009.
DNA Extraction Outline Purpose of DNA extraction

DNA Extraction Outline Purpose of DNA extraction
Microfluidic Hybrid Method for Simultaneous Screening and Optimization Validated with Crystallization of Membrane Proteins Liang Li, Cory Gerdts, Qiang.

Microfluidic Hybrid Method for Simultaneous Screening and Optimization Validated with Crystallization of Membrane Proteins Liang Li, Cory Gerdts, Qiang.
Peter Moeck, Department of Physics, www.physics.pdx.edu/~pmoeck Peter Moeck Dr. rer. nat. R.W. Chan: “one cannot easily get degrees in … crystallography”,

Peter Moeck, Department of Physics, Peter Moeck Dr. rer. nat. R.W. Chan: “one cannot easily get degrees in … crystallography”,
CHEMISTRY 59-320 ANALYTICAL CHEMISTRY Fall - 2010 Lecture 1.

CHEMISTRY ANALYTICAL CHEMISTRY Fall Lecture 1.
Barbara Mason, UCB Darren Edwards, Organon UK

Barbara Mason, UCB Darren Edwards, Organon UK
Collagen Fibrillogenesis Mary Beth Bird Mentor: Ramana Pidaparti, PhD. Mechanical Engineering Department July 23, 2007 Using Voltage to Optimize Protein.

Collagen Fibrillogenesis Mary Beth Bird Mentor: Ramana Pidaparti, PhD. Mechanical Engineering Department July 23, 2007 Using Voltage to Optimize Protein.
Goals  To find the ideal conditions to perform limited proteolysis  Most efficient trypsin:AP ratio  Buffer solution that optimizes trypsin activity.

Goals  To find the ideal conditions to perform limited proteolysis  Most efficient trypsin:AP ratio  Buffer solution that optimizes trypsin activity.
Future CAMD Workloads and their Implications for Computer System Design IEEE 6th Annual Workshop on Workload Characterization.

Future CAMD Workloads and their Implications for Computer System Design IEEE 6th Annual Workshop on Workload Characterization.
Lipinski’s rule of five

Lipinski’s rule of five
Results Initial chromatographic conditions [Figure 2a caption] for the separation of the degradation products of aspirin were problematic due to the long.

Results Initial chromatographic conditions [Figure 2a caption] for the separation of the degradation products of aspirin were problematic due to the long.
Astbury Centre, University of Leeds Jonathan Hadden Astbury Centre for Structural Molecular Biology University of Leeds Lessons Learned.

Astbury Centre, University of Leeds Jonathan Hadden Astbury Centre for Structural Molecular Biology University of Leeds Lessons Learned.
Two cases of chemometrics application in protein crystallography European Molecular Biology Laboratory (EMBL), Hamburg, Germany Andrey Bogomolov.

Two cases of chemometrics application in protein crystallography European Molecular Biology Laboratory (EMBL), Hamburg, Germany Andrey Bogomolov.
3D X-Ray Diffraction Microscopy Larry Margulies. 200 µm Metal Structures Heat Defor- mation 200 µm 5 µm Challenges: - Multiple lengthscales - Heterogeneities.

3D X-Ray Diffraction Microscopy Larry Margulies. 200 µm Metal Structures Heat Defor- mation 200 µm 5 µm Challenges: - Multiple lengthscales - Heterogeneities.
Alchemist TM II: Capacity, reproducibility and efficiency of an high-throughput crystallization screen preparation system. Craig Sterling Director of Business.

Alchemist TM II: Capacity, reproducibility and efficiency of an high-throughput crystallization screen preparation system. Craig Sterling Director of Business.
Douglas Instruments Microbatch seminar- slide 1 Should we be doing more crystallization by the microbatch method? Patrick Shaw Stewart Imperial College,

Douglas Instruments Microbatch seminar- slide 1 Should we be doing more crystallization by the microbatch method? Patrick Shaw Stewart Imperial College,
2004 PP&CW Optimization of protein expression and solubility Alternative and novel prokaryotic expression systems Eukaryotic expression systems Methods.

2004 PP&CW Optimization of protein expression and solubility Alternative and novel prokaryotic expression systems Eukaryotic expression systems Methods.

Similar presentations

Ads by Google