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A SEMI-AUTOMATED SYSTEM FOR NANOVOLUME PLUG-BASED CRYSTALLIZATION

PSI-2 Bottlenecks Meeting, Natcher Conference Center April 14-16, 2008. A SEMI-AUTOMATED SYSTEM FOR NANOVOLUME PLUG-BASED CRYSTALLIZATION . Cory Gerdts, Ph.D. April 15, 2008. ATCG3D is a Specialized PSI-2 Center Funded by the NIGMS and NCRR, Grant # U54-GM074961-03.

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A SEMI-AUTOMATED SYSTEM FOR NANOVOLUME PLUG-BASED CRYSTALLIZATION

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  1. PSI-2 Bottlenecks Meeting, Natcher Conference Center April 14-16, 2008 A SEMI-AUTOMATED SYSTEM FOR NANOVOLUME PLUG-BASED CRYSTALLIZATION Cory Gerdts, Ph.D. April 15, 2008 ATCG3D is a Specialized PSI-2 Center Funded by the NIGMS and NCRR, Grant # U54-GM074961-03 Slide prepared by Lance Stewart

  2. Outline • Technology Background • Project Update • Upcoming Improvements Slide prepared by Cory Gerdts

  3. Droplets (Plugs) in Microfluidic Channels fluorocarbon Langmuir (2003) Appl. Phys. Lett. (2003) Anal. Chim. Acta (2004) Philos. T. Roy. Soc. A (2004) H. Song, J. D. Tice, R. F. Ismagilov Angew. Chem.-Int. Edit. 2003, 42, 768 Slide prepared by Cory Gerdts

  4. Labcard The Microfluidic Protein Crystallization System: (MPCS) A Three Component System Slide prepared by Cory Gerdts

  5. Crystallization in Plugs Slide prepared by Cory Gerdts

  6. 200 mm Gradient and Sparse Matrix Screening Formation of Fine Gradients Zheng B.; Roach L. S.; R. F. Ismagilov J. Am. Chem. Soc. 2003, 125: 11170-11171. Sparse Matrix Screening with Pre-Formed Cartridges Pre-formed Cartridge - ~10 nL plugs Zheng, B.; Ismagilov, R.F. Angew. Chem. 2005, 117: 2576-2579. Slide prepared by Cory Gerdts

  7. Hybrid Method: Sparse Matrix + Gradient Screening + L. Li, D. Mustafi, Q. Fu, V. Tereshko, D.L. Chen, J.D. Tice, R.F. Ismagilov PNAS, 2006,103, 19243. Slide prepared by Cory Gerdts

  8. Hybrid Method Slide prepared by Cory Gerdts

  9. Outline • Technology Background • Project Update • Upcoming Improvements Slide prepared by Cory Gerdts

  10. MPCS Development Goals • Labcard • Flat • Thin • Rigid • Low Dead Volume Macro-Micro interface • Injection Molded Parts (low cost) • One-time Use • Plastic Material Properties: • Transparent • Low Birefringence • X-ray Transmissive • Low Surface Energy (hydrophobic/fluorophilic) • Pump System • Constant Flow at Low Flow Rates • Ability to Form Smooth Gradients • No Delay/Response Time • Dissemination • Disseminate Technology to Center and Community • Make Technology Available Slide prepared by Cory Gerdts

  11. MPCS - Beta

  12. Multiple-Pronged Approach: 2 Functional Labcards SBS Formatted Plate Slide prepared by Cory Gerdts

  13. MPCS Advantages/Features: Diffraction-Ready Crystals ribose-phosphate pyrophosphokinase methionine-R- sulfoxide reductase Reaction Center Acyl-CoA hydrolase Fatty Acid Amide Hydrolase Myoglobin Porin TDP Slide prepared by Cory Gerdts

  14. Protein Crystal Extraction Methionine-R-sulfoxide Reductase data set: 1.7 Å resolution Slide prepared by Cory Gerdts

  15. MPCS Advantages/Features: In Situ Diffraction Data merged from 3 crystals: 1.9 Å resolution Slide prepared by Cory Gerdts

  16. pH 6 pH 7 pH 8 MPCS Advantages/Features: Optimization • Applications of small volume fine gradient screening: • Optimize buffer system, precipitant concentration, and pH • Identify optimal or tolerable doses of additives to known crystallization conditions • * Cryoprotectants * Ligands • * Other Protein Partners * Other DNA Partners • * Detergents * Heavy Atoms Improve efficiency: *Mutants * Ligands *DNA complexes • Reduce protein preparatiion requirements for screening Slide prepared by Cory Gerdts

  17. Results of a Smooth Gradient

  18. MPCS Advantages/Features: No Dead Volume • Protein is aspirate into a piece of Teflon tubing on a syringe that is back-filled with the fluorocarbon (FC) oil. Every nL of protein gets displaced into the system. • 4 mL of protein is enough for ~800 experiments. As little as 0.5 mL protein can be aspirated easily. Slide prepared by Cory Gerdts

  19. MPCS Advantages/Features: Microfluidic Seeding Microcrystals from a previous trial can be used to performmicrofluidic seeding in the MPCS 1. Microcrystals are aspirated into a Teflon tube 2. The microcrystal-containing solution is used as one of the aqueous streams in the 3+1 mixer. 3. The Labcard is filled with plugs that each contain one or a few small microcrystals. • Microfluidic seeding can be used to: • Optimize a protein that nucleates too much, or • Induce crystallization in a mutant, ligand, DNA, etc. screen by crushing up a crystal from a known condition or from the wild type version Slide prepared by Cory Gerdts

  20. Outline • Technology Background • Project Update • Upcoming Improvements Slide prepared by Cory Gerdts

  21. Microcapillary Crystallization: Next Steps • Development of more user friendly full instrument • Improved connections (hybrid in plastic Labcards) • Automatic Aspirator • Improved pumps • Further Dissemination

  22. Microcapillary Protein Crystallization Workshop Counterdiffusion Microcapillary Crystallization Micropipet / Microcapillary Handling and Crystallization Date: June 6-7, 2008 Location: deCODE biostructures Emerald BioSystems 2501 Davey Road Woodridge, IL 60517 Plug-Based Microcapillary Crystallization Slide prepared by Cory Gerdts and Lance Stewart

  23. Acknowledgements University of Chicago: • RustemIsmagilov • Liang Li • George Sawicki • DebarshiMustafi • ValentinaTerechko • Qiang Fu • Wenbin Du • Anna Selezneva • Microcapillary Crystallization deCODE: • Lance Stewart (PI) • Peter Nollert • Mark Elliot • Yiping Xia The Scripps Research Institute: • Raymond Stevens (Co-PI) • Peter Kuhn • Peter Clark • Vadim Cherezov • Joe Ng For more information: Cory Gerdts Phone: 630-783-4691 Email: cgerdts@ emeraldbiosystems.com • ATCG3D Funding NIGMS / NCRR U54-GM074961

  24. Methionine-R-sulfoxide Reductase data set Space group – P1 (Triclinic) Unit cell – a = 42.00, b = 45.17, c = 45.40 Angles – a = 88.4, b = 83.7, g = 69.1 Scaled from 50 – 1.7 Completeness – 95.3 (87.4) Rmerge – 6.8% (42.3) I/sI – 23.1 (2.2) # of reflections -118,181 (total), 32,539 (unique) Redundancy 3.6 (3.3)

  25. Counterdiffusion Confined Geometry Microcapillary Crystallization • Topas COC plastic microfluidic crystallization Greiner Bio-One labcard • Individual Lysozyme Crystals grown in microcapillary channels by counterdiffision methods • In situ X-ray diffraction on individual crystals reveals different diffraction quality Prototype Design Prototype Prototype Greiner Bio-One Commercialization of Confined Geometry Counterdiffusion Labcard Slide prepared by Lance Stewart from data of Joe Ng and Peter Kuhn

  26. Summary of MPCS Advantages and Features • USABLE soluble AND membrane • protein crystals grow in ~10 nL plugs • In situ diffraction for both data • acquisition and crystal quality screening • Increased efficiency from preparing • decreasing amounts of protein • On-chip formulation for fine granularity screening for “narrow • crystallization slots” • ~800 experiments per card • No dead volume – every nL gets used • Large Phase Space can be investigated with the Hybrid Method using • preformed cartridges • Microfluidic seeding Slide prepared by Cory Gerdts

  27. Nanovolume Microcapillary Crystallization with In Situ X-ray Imaging • Emerging Technologies • Microfluidic Device Production (PDMS, Plastic, Glass) • Microfluidic Pumping Systems (Various) • Novel Microfluidic Circuitry and Methods for Protein Crystallization (Seeding, Gradient, Random Sparse, Microbatch, Counterdiffusion) • Opportunity • Efficient Exploration of Crystallization Space per Unit Protein • Integration of Crystallization with X-ray Data Collection (In Situ) • Efficiency Gain • Increased Crystallization Success per Unit of Protein • Improved In situ Diffraction Quality Screening (true measure of a crystal) • Improved Inventory Staging of Crystals for X-ray Data Collection Slide prepared by Lance Stewart

  28. Simple 4-Microsyringe Pump System and “Microplugger” Control Software and Labcard Prototype Emerald BioSystems is offering commercial systems as of July 2007 pH Gradient Test, ~10nl Plugs Microplugger™ Control Software In Situ X-ray Diffraction of Protein Crystals in Labcards Micro-Syringe Pump System Labcard Prototype Slide prepared by Lance Stewart

  29. ATCG3D Technology Focus Areas • Compact Light Source for Tunable X-ray Diffraction Data Collection in a Laboratory Setting • Nanovolume Microcapillary Crystallization with In Situ X-ray Imaging • Computer Aided Protein Construct Engineering and Synthetic Gene Design Slide prepared by Lance Stewart

  30. DETECT-X Imaging of Protein Crystals in Microfluidic Devices • DETECT-X Imager used in conjunction with Millipol software • High signal to noise imaging of crystals • Crystal orientation imaging • Potential to Enable Efficient Complete in situ X-ray diffraction Data Set Collection Emerald BioSystems, Commercial Launch of DETECT-X, Q2 2007 W. Kaminski, Millipol Software Norgren Systems LLC Hardware Engineering Slide prepared by Lance Stewart with data from Peter Nollert

  31. Future Directions Gene-to-3D in 3 Days ? • Starting from Oligonucleotides • PCR based gene production / transcription  mRNA, 8 h • Cell free translation for protein production, 16 h • Affinity purification of protein, 4 h • Nanovolume microcapillary labcard crystallization, 20 h • In situ X-ray diffraction data collection, 6 h • Structure Determination 16 h • TOTAL ~70 h 3D in 3 Day Challenge Slide prepared by Lance Stewart

  32. Acknowledgements • ATCG3D PI Collaborators • Peter Kuhn and Raymond Stevens (Co-PIs, The Scripps Research Institute) • Synthetic Gene Design Software • John Walchli (deCODE) • Alex Burgin (deCODE) • Mark Mixon (deCODE) • Don Lorimer (deCODE) • Corporate Collaborators • Peter Nollert, Emerald BioSystems • Lawrence Kuo, J&JPRD • Masaki Madono, Cell Free Sciences • Synthetic Gene Design Wet Lab Work • Don Lorimer (deCODE) • Ellen Wallace (deCODE) • Amy Raymond (deCODE) • Adrienne Metz (deCODE) • Rena Grice (deCODE) • PSI-2 / SG Collaborators • James Love, NYCOMPS • Dmitriy Vinarov, CESG, Univ. of Wisconsin • Mark Sullivan, University of Rochester • Alexei Brooun, TSRI now at Pfizer • Yaeta Endo, Ehime University • PSI-2 Grant Funding • ATCG3D Funding NIGMS / NCRR U54-GM074961 • Contract Funding • SSGCID Funding from NIAID HHSN272200700057C • SSGCID (NIAID) Collaborators • Peter Myler, Seattle Biomedical Research Institute • Wes Van Voorhis, University of Washington Slide prepared by Lance Stewart

  33. Microcapillary Protein Crystallization Workshop Counterdiffusion Microcapillary Crystallization Micropipet / Microcapillary Handling and Crystallization Date: June 6-7, 2008 Location: deCODE biostructures /Emerald BioSystems 2501 Davey Road Woodridge, IL 60517 Plug-Based Microcapillary Crystallization www.MPCW2008.org Slide prepared by Cory Gerdts

  34. MPCS Advantages/Features: Membrane Proteins • Membrane protein crystallization is done using Teflon capillaries and PDMS 3+1 mixers. • Storage capillary controls evaporation Slide prepared by Cory Gerdts

  35. ATCG3D

  36. Becoming a Beta User of theMicrocapillary Protein Crystallization System • Beta User obtains complete Microcapillary Protein Crystallization System • Training: #1: training of Beta User scientists at Emerald site (Bainbridge Island, WA) • Installation: complete installation of the Microcapillary Protein Crystallization System in Beta User laboratory, validation and QC • Training #2: training of Beta User scientists at Beta User site • Cost: $24k for system, $19k for annual technology access license • Emerald solicits feedback to improve system performance • Beta User agrees not to reverse engineer system, resulting IP owned by Emerald, co-authorship of publication

  37. Nanovolume Protein Crystallization: PDMS 3+1 Mixer and Microcapillary Holder PDMS 3+1 Mixer 0.45 meter of Teflon capillary stored in glass capillary 1 meter of tubing, ~1000 membrane protein crystalliization trials Li, L., Mustafi, D., Fu, Q., Tereshko, V., Chen, D.L., Tice, J.D., Ismagilov, R.F. PNAS 2006, 103: 19243 Slide prepared by Cory Gerdts

  38. Only Precipitation and Crystal Clusters in Optimized Vapor Diffusion Experiments Microfluidic Separation of Nucleation and Crystal Growth Yields Single Crystals Crystallization of Oligoendopeptidase Fby Microfluidic Seeding Target: apc36224 selected by MCSG but unsolved

  39. Oligoendopeptidase F(B. stearothermophilus) Structure • Oligoendopeptidase F • apc36224 • 3.1 Å resolution • Space group: P3121; Unit Cell • Parameters a=b 119.50 c=248.90 • R-factor = 0.196, R-free = 0.248 • Solvent Content: ~70%; • PDB Accession #s 2H1N and 2H1J • Zn++ Metallopeptidase family M3 • Structural similarity to: • human testicular Angiotensin-converting enzyme, 1O86 • E.coli Dipeptidyl Carboxypeptidase Dcp, 1Y79 • Neurolysin, 1I1I • Pyrococcus furiosus carboxypeptidase, 1K9X • ATCG3D and MCSG collaboration Time-Controlled Microfluidic Seeding in nL-Volume Droplets To Separate Nucleation and Growth Stages of Protein Crystallization Gerdts, C.J., Tereshko, V., Yadav, M.K., Dementieva, I., Collart, F., Joachimiak, A., Stevens, R.C., Kuhn, P., Kossiakoff, A., and Ismagilov, R.F. Angew. Chem. Int. Ed. 2006 45: 8156-8160

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