1 / 35

Crystallization Methods for Structural Genomics @ AFMB

Crystallization Methods for Structural Genomics @ AFMB. Steps towards a crystal structure ranked by difficulty (bottlenecks). Protein production & purification. Production of diffracting crystals. Structure determination. Reasons to use a crystallization robot?.

tejano
Download Presentation

Crystallization Methods for Structural Genomics @ AFMB

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Crystallization Methods for Structural Genomics @ AFMB

  2. Steps towards a crystal structure ranked by difficulty (bottlenecks) Protein production & purification Production of diffracting crystals Structure determination

  3. Reasons to use a crystallization robot? Why cars are made by robots? •Automatization: -gain time -manpower (money) -no mistakes •Miniaturization: save precious material •More hits !!!

  4. •Horizontal organization •Automatization (robots) •Miniaturization •Independent from lab’s targeted projects Analysis Structure Crystallization Protein production and Purification ORF sub-cloning and expression

  5. PCR Overexpression X-ray data Gateway Solubility 3D-Structures Colony screening Crystallization Transformation Crystals First round Second round

  6. SDS-PAGE IEF (iso-electric focusing) Protein characterization • Mass spectra Mass, substitution Met->Se-Met • Circular Dichroïsm • Dynamic Light Scattering (DLS)

  7. Screening of crystallization conditions  sparse matrix MDL 1 et 2 100 conditions http://www.moleculardimensions.com/ Stura 48 conditions http://www.moleculardimensions.com/ Références: 1. Stura E.A. (1999) In Protein Crystallization: Techniques, Strategies and Tips. A laboratory manual. (Bergfors T. ed.) International University Line. Pp113-124. Wizard 96 conditions http://www.emeraldbiostructures.com/ Clear 96 conditions http://www.moleculardimensions.com/ Références  Dauter, Z, Dauter, M & Rajashankar, K. R. (2000), Acta Cryst. D56, 232-237. Zetazol 72 conditions http://www.moleculardimensions.com/ Références Riès-Kautt M, Ducruix A. Methods Enzymol (1997), 276:23-59. 412 conditions of crystallization

  8. Crystallization automatization: robots • Institute made robot • Hanging drop1.5mL+ 1.5mL • Limbro plates • Plates greasing

  9. Screening of crystallization conditions • 412 conditions • - 3 concentrations => 1236 drops Storage rack for kits Greiner plates 288 shelves

  10. TECAN ROBOT Micro-drops + plate preparation CARTESIAN ROBOT Nano-drops

  11. Crystallization automatization: robots • TECAN robot (http://www.tecan.com) • 1.5mL+ 1.5mL • Greiner • Controlled atmosphere • 45 min • Protein amount = 20mg

  12. Use of the TECAN robot (1/9/2001-31/3/2002) • • A close cabinet has been added • • easy to program • Problems: • “Low volume” needles, slow • Dispense by contact: lost drops ≈ 0-20% (precipitant and • protein dependant) • NOW: prepare the greiner plate

  13. CARTESIAN (http://www.cartesiantech.com)

  14. master controller nQUAD valve stage stepper motor syringe stepper motor Technology synQUAD • Valves, seringes, are synchronised • dispense “in the fly” ou “step by step” • Very fast

  15. Sitting drop • Greiner 96 • Protein volume / plate: 36 - 120 ml • Drops volumes • (100 à 500 nL protein • + 100 à 500 nL precipitant) • Controled athmosphere • Time: 15’ / 288 drops Cartesian inc. RobotMulti-plate dispensing

  16. Protein dispensing Drops dispensing Reservoir dispensing Tips washing

  17. Nanodrops Plus : - small amounts of protein - non-contact dispensing Example : - 412 conditions - Protein: 3 concentrations (5, 10, et 15 mg/ml) 1236 drops =1,5 mg de protéine Technical constraints: - Fast vapor diffusion => controlled humidity (80-90% saturation) => fast dispensing - Précision => contact protein+precipitant => centering

  18. Semi-automated visualization No crystal recognition yet

  19. Storage and visualization cabinet for 600-1200 plates • Visualization: 1plate/min• Crystal detection• Bar code reading• LIMS interfaced

  20. Results with nano-drops Flat bottom Greiner Lysozyme Target 87 Round bottom Greiner

  21. Results

  22. Refinement of the crystallization conditions 4 stock solutions, dispensed by the Tecan as a 8 x 8 matrix - Time : 25 minutes [precipitant] pH

  23. Refinement of the crystallizations: Nano-drops ( matrix 8x8) – Cartesian Robot [Precipitant] pH

  24. Diffraction Queen honey bee Pheromone Binding Protein 6.8 Å 3.4 Å 2.3 Å 1.7 Å

  25. Queen bee Pheromone Binding ProteinInitial drop size 300nL 3 l 300nLX-ray source Rotating anode ID14-EH1 ID14-EH2l (Å) 1.5418 0.933 0.933Resolution 30.0-2.9 28.4-2.0 28.4-1.6ranges (Å) 3 .06/2.90 2.11-2.0 1.66-1.6Rsym (%) 8.4/26.7 3.9/9.5 3.6/26.7I/I) 5.0/2.4 7.2/3.1 10.2/2.6Completeness 99.8/99.8 99.1/99.1 99.7/99.7Multiplicity 4.9/5.0 5.4/5.5 3.8/3.8

  26. Cockroach Pheromone Binding Protein MAD data set at the Se edge BM14 (ESRF) Peak : 0.97978 Å Resolution : 20 – 1.7 Å Space group: P1 Cell : 43.3 44.92 45.56 Å 118.1° 93.78° 107.15° Completeness : 95.7% Rmerge : 4.8% Rano : 6.8% I/sI : 7.4 Multiplicity : 6.0 observations : 174 860 reflections : 29347

  27. Present procedure: 1/screening Kit solutions Nikon Biotom TECAN Cartesian 2/ Refinement: 4 hand-made solutions

  28. Future procedure: Automated Loop mounting Crystal carousel 1/screening Kit solutions SR twister Plate hotel Plate sealing Cartesian or else Biotom cabinet TECAN Pipetting robot: 4 refinement solutions • Crystal recognition • LIMS • Refinement design

  29. Project of NUNC high density (1536 independent wells) plate

  30. ReferencesStructural Genomic of Bacterial Proteins at Lab Scale: new strategies for proteins expression and crystallization,Renaud Vincentelli, Christophe Bignon, Arnaud Gruez, Gerlind Sulzenbacher, Mariella Tegoni, Valérie Campanacci and Christian Cambillau, Accounts Chem. Res., in press. A Medium Throughput Crystallization Approach. Gerlind Sulzenbacher, Arnaud Gruez, Véronique Roig-Zamboni, Silvia Spinelli, Christel Valencia, Fabienne Pagot, Renaud Vincentelli, Christophe Bignon, Aurelia Salamoni, Sacha Grisel, Damien Maurin, Céline Huyghe, Kent Johansson, Alice Grassick, Alain Roussel, Yves Bourne, Sophie Perrier, Linda Miallau, Philippe Cantau, Eric Blanc, Michel Genevois, Alain Grossi, André Zenatti, Valérie Campanacci, & Christian Cambillau. Acta Crystallogr. D D58, 2109-2115 (2002)

  31. TROUBLESHOOTING• A very common mistake is to place plates upsidedown; you can distroy your 8 tips. Check carefully.

  32. TROUBLESHOOTING• Tips are fragile an can be easily broken. • Be careful while manipulating.

  33. Valérie Campanacci: ASG/SPINE manager Stephane Canaan: TB manager Aline Desmyter: MePNet manager Christel Valencia: ASG manager Renaud Vincentelli: new developments Corinne Rancurel: bioinformatics Arnaud Gruez: X-ray, Cartesian Aurelia Salamoni: mol biol, biochem Sacha Grisel: mol biol, biochem Fabienne Pagot: crystallization screen/robots Damien Maurin, mol biol, biochem Cécile Durousseau, mol biol, biochem Frédéric Frassinetti, mol biol, biochem Lorena Scappuccini, crystallization, biochem Marie-eve Gravière, mol biol, biochem Céline Huyghe: mol biol, biochem Fabienne Tocque: mol biol, biochem Nicolas Brémont: mol biol, biochem Manuela Dolzan: crystallization,X-ray Christophe Bignon: Mol-Bio manager Véronique Zamboni: crystal refinement Silvia Spinelli: crystal refinement, structure Gerlind Sulzenbacher: X-ray, robots Mariella Tegoni: redox, enzymology Yves Bourne: head TB Bruno Canard: head SPINE Christian Cambillau: SG projects manager Xray involved: several people from the lab SG TEAM @ AFMB http://afmb.cnrs-mrs.fr/stgen/

More Related