PROTEIN CRYSTALLIZATION

Transcription

1 PROTEIN CRYSTALLIZATION TECHNIQUES, STRATEGIES, AND TIP S A Laboratory Manua l TERESE M. BERGFORS

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3 Preface List of Contributors v xvii 1. A Bit of Advice on Crystallizing Proteins 3 Alexander McPherson 2. Crystallization Methods 7 Torsten Unge 2.1. The Typical Vapor Diffusion Experiment Hanging Drops Sitting Drops Sandwich Drops Reverse Vapor Diffusion ph Gradient Vapor Diffusion Practical Tips for Vapor Diffusion Other Methods Dialysis Batch Techniques Microbatch Summary 1 4 Lab Experiment : Crystallization of Hen Egg White Lyzosome by Two Different Methods 1 6 Experiment 2.1. Vapor diffusion hanging drop technique 1 6 Experiment 2.2. Crystallization by the batch method Protein Samples 19 Terese Bergfors 3.1. Lyophilization Ammonium Sulfate Precipitation Keep Purification Batches Separate Characterize the Protein 22

4 3.5. Storage of Protein Be Gentle Keep Good Records Learn All about the Protein Dynamic Light Scattering 27 Terese Bergfors 4.1. What Does DLS Measure? Why Use DLS to Measure Size Homogeneity? Performing and Evaluating DLS Measurements on Your Protein 3 1 Instrument 3 1 Part 1 : Sample Preparation 32 Part 2: Further Testing of Protein Solvent Conditions with DLS Recommendations for Handling Your Protein Base d on Its DLS Profile If Your Protein Is MONOMODAL If Your Protein Is Not MONOMODAL Summary Precipitants 3 9 Terese Bergfors 5.1. Which Precipitants to Use? Which Concentration Range to Use? Quick Protocol for Determining the Precipitatio n Point of a Protein Grid Screens Comments 4 6 A 5.1. Precipitants for Protein Crystallization Buffers and ph 5 1 Terese Bergfors and Kerstin Fridborg 6.1. Buffers Buffer Concentration for the Protein Solution Buffer Concentration for the Drops Choice of Buffer ph Initial Choice of ph Range for Screening Optimization of ph 57 A 6.1. Tables of Buffers Versus Number o f Successful Crystallizations in the BMCD The most commonly used buffers in crystallizatio n experiments sorted by frequency of occurrence 58

5 2. Buffers available from Hampton Research and thei r occurrence in the BMCD Inorganic buffers versus number of crystallizations Good's biological buffers versus number of crystallizations Temperature 63 Lesley Lloyd Haire 7.1. Temperature as a Crystallization Parameter Recommendations When Working at 4 C Testing the Effects of Temperature Crystallization by Temperature Gradients Crystallization Strategies 69 Terese Bergfors 8.1. The Problem Types of Screens : Pros and Cons Crystallization Strategy Is More Than a Choic e of Screening Method Strategy 1 : A Flexible Sparse Matrix Screen 77 Johan Philip Zeelen 9.1. The Protein Sample Protein Concentration and Starting Buffer Crystallization with a Fast-Screening Protocol The Initial Screen Preparing the Initial Screen Adjusted Screen Optimize the Crystallization Conditions Optimization Step Optimization Step If the Crystals Are Not Good Enough When Nucleation Is Not Evident 84 A 9.1. Tables for the "Flexible Sparse Matrix Screen" Composition of the 48 well solutions of the initial screen Stock solutions and pipetting schem e for the initial screen Example of an optimization experiment Additives Strategy 2 : An Alternative to Sparse Matrix Screens 9 1 Madeleine Ries-Kautt Introduction 93

6 10.2. Estimation of the Net Charge Choice of the ph for Crystallization Buffers The Initial Test Crystallization of Proteins Having a Positive Net Charge Range of Salt Concentration to Use Crystallization Crystallization of Proteins Having a Negative Net Charge Range of Salt Concentration to Use Crystallization Crystallization of Proteins Having a Net Charge of about Zero Range of Concentration to Use Crystallization Optimizing the Nucleation Rate The Phase Diagram Optimal Conditions at Moderate Concentrations of the Crystallizing Agent 10 5 A10.1. Spread Sheet for the Calculation of Protei n Net Charge versus ph 10 7 A10.2. Equations to Calculate a Protein Net Charge 10 8 A10.3. Miscibility Curves for Organic Crystallizin g Agents and Various Salts Strategy 3: Reverse Screening 11 1 Enrico A. Stura Specificity of Precipitants and Buffers 11 3 Experiment Monomethyl PEG (MPEG) crystallization of lysozyme Search at a High Degree of Supersaturation Screening Solubility Evaluation 11 7 Experiment Solubility screening Additive Screening Use of Additives Magic Solution Heavy Atom Soaking and Cryo-Crystallography 120 Experiment Solubility screening applied to heav y atom soaking Some Additional Tips in Preparing Heavy Atom Derivatives Summary 12 2 A Tables for a PEG Screen and Common Crystallization Additives 123

7 1. PEG screen Commonly used additives in the crystallization of biological macromolecules Strategy 4: Imperial College Grid Screen 12 5 Lesley Lloyd Haire The Imperial College Screen The Imperial College Supplemental Screen Interpretation of the Crystallization Drop Results 13 1 Johan Philip Zeele n Result Interpretation and Type of Screen The Stereomicroscope Examination of the Crystallization Experiments Seeding 13 9 Enrico A. Stura Streak Seeding Preparing Seed Stock 144 Experiment Streak seeding and seed transfer Diagnostic Uses of Seeding Seeding and Sitting Drops Optimization Seeding as a Screening Technique Heterogeneous Seeding 14 8 Experiment Epitaxial jumps Recognizing an Epitaxial Jump Cross Seeding Summary 15 1 A Recommendations for Streak Seeding and Seed Transfer Macroseeding: A Real-Life Success Story 155 Sherry L. Mowbray Why Do Macroseeding? How to Macroseed 158 Step 1. Obtain seeds 15 8 Step 2. Determine the solubility of the crystals 158 Step 3. Generate a wash protocol 159 Step 4. Establish appropriate equilibrium conditions 160 Step 5. Analyze results Common Problems/Causes Too Many Crystals Dissolving Seeds 161

8 Poor Crystals Oils for Crystals 163 Naomi E. Chayen The Rationale for Crystallization under Oil The Microbatch Technique Setting up Crystallization Trials by Microbatch Crystallization of Membrane Proteins in Oil Additional Benefits of Oil Limitations of Crystallizing under Oil Harvesting Microbatch Crystals The Effect of Different Oils on Microbatch Use of Different Oils in Screening Use of Oils in Optimization Control of Nucleation Effect of Surface Contact on Nucleation The Use of Oil for Controlling the Rate of Vapor Diffusion Trials Summary 174 Lab Exercises with Oils 175 Exercise Setting up microbatch trials 17 5 Exercise 16.2, Filtration experiments 17 7 Exercise Containerless crystallization 17 8 Exercise Insertion of oil barrier to slow down vapor diffusion experiments Crystallization for Cryo-Data Collection 181 Elspeth F Garman Introduction Protocol for Finding Crystallization Conditions fo r Cryo-Crystallography Laboratory Exercise for Determining Cryo-Protectant Solutions Mounting Crystals for Room Temperature Data Collection General Procedure for Mounting a Crystal 190 A Glycerol Concentrations Required t o Cryo-Protect CRYSTAL SCREEN I solutions Crystallization of Membrane Proteins 197 JeffAbramson and So Iwata Principles of Membrane Protein Crystallization Practical Approach for Membrane Protein Crystallization Sample Preparation Selection of Detergent 202

9 Purification Protein Concentration and Detergent Exchange Crystallization Selection of the Detergent Crystallization Setups Precipitants Temperature Screening Kits Optimization One Last Piece of Advice : Appearances Can Be Deceiving! 21 1 Alex Camero n A-Z Tips 21 5 APPENDICES 24 9 Appendix Al. Good-to-Have Gizmos 25 1 A1.l. Temperature Logger for the Crystallization Room 25 1 A1.2. Positive Displacement Pipettes 25 1 A1.3. Microelctrode for Measuring ph in the Drop Reservoir 25 2 A1.4. Buffer Exchange for Small Volumes (<250 ml) of Proteins 25 3 A1.5. Vortex Mixer Adapted for 24-Well Tissue Culture Plates 25 5 A1.6. Automated Grease Dispenser 25 5 Appendix A2. Supplies for Crystallization and Suggested Sources 25 7 Appendix A3. Suppliers' Addresses 26 1 Appendix A4. Useful Websites 269 Appendix AS. Commercially Available Screens 273 A5.1. Crystal Screen I 27 3 A5.2. Crystal Screen II 276 A5.3. Additives Screens 1, 2, and A5.4. Wizard I 282 A5.5. Wizard II 285 A5.6. Cryo I 288 A5.7. Cryo II 29 1 A5.8. Structure Screen A5.9. Structure Screen Index 301