Protein Crystallization: Techniques, Strategies, and Tips. A Laboratory Manual


IUL Biotechnology Series, 1
by Terese M. Bergfors (Editor)

Edition: First

Book Details:

  • Series: IUL Biotechnology Series
  • Volume: 1
  • Binding: Hardcover
  • Pages: 306
  • Dimensions (in inches): 1.25 x 9.75 x 6.50
  • Publisher: International University Line
  • Publication Date: October 15, 2002 (Second Print)
  • ISBN: 0-9636817-5-3
  • Price: $69.95

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Techniques, Strategies, and Tips

A Laboratory Manual
edited by

Preface     v
List of Contributors     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     9
            2.1.1. Hanging Drops     10
            2.1.2. Sitting Drops     10
            2.1.3. Sandwich Drops     11
            2.1.4. Reverse Vapor Diffusion     11
            2.1.5. pH Gradient Vapor Diffusion     11
            2.1.6. Practical Tips for Vapor Diffusion     12
     2.2. Other Methods     12
            2.2.1. Dialysis     13
            2.2.2. Batch Techniques     14
            2.2.3. Microbatch     14
     2.3. Summary     14
     Lab Experiment: Crystallization of Hen Egg White Lyzosome by Two Different Methods     16
            Experiment 2.1. Vapor diffusion hanging drop technique     16
            Experiment 2.2. Crystallization by the batch method     17
3. Protein Samples     19
Terese Bergfors
     3.1. Lyophilization     21
     3.2. Ammonium Sulfate Precipitation     22
     3.3. Keep Purification Batches Separate     22
     3.4. Characterize the Protein     22
     3.5. Storage of Protein     23
     3.6. Be Gentle     24
     3.7. Keep Good Records     24
     3.8. Learn All about the Protein     25
4. Dynamic Light Scattering     27
Terese Bergfors
     4.1. What Does DLS Measure?     29
     4.2. Why Use DLS to Measure Size Homogeneity?     29
     4.3. Performing and Evaluating DLS Measurements on Your Protein     31
            Instrument     31
                  Part 1: Sample Preparation     32
                  Part 2: Further Testing of Protein Solvent Conditions with DLS     35
     4.4. Recommendations for Handling Your Protein Based on Its DLS Profile     36
            4.4.1. If Your Protein Is MONOMODAL     36
            4.4.2. If Your Protein Is Not MONOMODAL     37
     4.5. Summary     37
5. Precipitants     39
Terese Bergfors
     5.1. Which Precipitants to Use?     41
     5.2. Which Concentration Range to Use?     42
     5.3. Quick Protocol for Determining the Precipitation Point of a Protein     44
     5.4. Grid Screens     45
     5.5. Comments     46
     A 5.1. Precipitants for Protein Crystallization     48
6. Buffers and pH     51
Terese Bergfors and Kerstin Fridborg
     6.1. Buffers     53
            6.1.1. Buffer Concentration for the Protein Solution     53
            6.1.2. Buffer Concentration for the Drops     53
            6.1.3. Choice of Buffer     54
     6.2. pH     56
            6.2.1. Initial Choice of pH Range for Screening     56
            6.2.2. Optimization of pH     57
     A 6.1. Tables of Buffers Versus Number of Successful Crystallizations in the BMCD     58
                1. The most commonly used buffers in crystallization experiments sorted by frequency of occurrence     58
                2. Buffers available from Hampton Research and their occurrence in the BMCD     59
                3. Inorganic buffers versus number of crystallizations     59
                4. Good’s biological buffers versus number of crystallizations     60
7. Temperature     63
Lesley Lloyd Haire
     7.1. Temperature as a Crystallization Parameter     65
     7.2. Recommendations When Working at 4ºC     66
     7.3. Testing the Effects of Temperature     66
     7.4. Crystallization by Temperature Gradients     67
8. Crystallization Strategies     69
Terese Bergfors
     8.1. The Problem     71
     8.2. Types of Screens: Pros and Cons     72
     8.3. Crystallization Strategy Is More Than a Choice of Screening Method     74
9. Strategy 1: A Flexible Sparse Matrix Screen     77
Johan Philip Zeelen
     9.1. The Protein Sample     79
            9.1.1. Protein Concentration and Starting Buffer     79
     9.2. Crystallization with a Fast-Screening Protocol     80
            9.2.1. The Initial Screen     80
            9.2.2. Preparing the Initial Screen     81
            9.2.3. Adjusted Screen     82
     9.3. Optimize the Crystallization Conditions     82
            9.3.1. Optimization Step 1     82
            9.3.2. Optimization Step 2     83
            9.3.3. If the Crystals Are Not Good Enough     83
            9.3.4. When Nucleation Is Not Evident     84
     A 9.1. Tables for the “Flexible Sparse Matrix Screen”     84
                1. Composition of the 48 well solutions of the initial screen     84
                2. Stock solutions and pipetting scheme for the initial screen     86
                3. Example of an optimization experiment     89
                4. Additives     90
10. Strategy 2: An Alternative to Sparse Matrix Screens     91
Madeleine Riès-Kautt
     10.1. Introduction     93
     10.2. Estimation of the Net Charge     94
     10.3. Choice of the pH for Crystallization     95
              10.3.1. Buffers     96
              10.3.2. The Initial Test     96
     10.4. Crystallization of Proteins Having a Positive Net Charge     97
              10.4.1. Range of Salt Concentration to Use     97
              10.4.2. Crystallization     98
     10.5. Crystallization of Proteins Having a Negative Net Charge     98
              10.5.1. Range of Salt Concentration to Use     99
              10.5.2. Crystallization     99
     10.6. Crystallization of Proteins Having a Net Charge of about Zero     100
              10.6.1. Range of Concentration to Use     100
              10.6.2. Crystallization     101
     10.7. Optimizing the Nucleation Rate     101
              10.7.1. The Phase Diagram     101
              10.7.2. Optimal Conditions at Moderate Concentrations of the Crystallizing Agent     105
     A10.1. Spread Sheet for the Calculation of Protein Net Charge versus pH     107
     A10.2. Equations to Calculate a Protein Net Charge     108
     A10.3. Miscibility Curves for Organic Crystallizing Agents and Various Salts     109
11. Strategy 3: Reverse Screening     111
Enrico A. Stura
     11.1. Specificity of Precipitants and Buffers     113
              Experiment 11.1. Monomethyl PEG (MPEG) crystallization of lysozyme     114
     11.2. Search at a High Degree of Supersaturation     115
     11.3. Screening     116
     11.4. Solubility Evaluation     117
              Experiment 11.2. Solubility screening     117
     11.5. Additive Screening     118
              11.5.1. Use of Additives     119
              11.5.2. Magic Solution     120
     11.6. Heavy Atom Soaking and Cryo-Crystallography     120
              Experiment 11.3. Solubility screening applied to heavy atom soaking     120
              11.6.1. Some Additional Tips in Preparing Heavy Atom Derivatives     121
     11.7. Summary     122
     A 11.1. Tables for a PEG Screen and Common Crystallization Additives     123
                  1. PEG screen     123
                  2. Commonly used additives in the crystallization of biological macromolecules     124
12. Strategy 4: Imperial College Grid Screen     125
Lesley Lloyd Haire
     12.1. The Imperial College Screen     127
     12.2. The Imperial College Supplemental Screen     128
13. Interpretation of the Crystallization Drop Results     131
Johan Philip Zeelen
     13.1. Result Interpretation and Type of Screen     133
              13.1.1. The Stereomicroscope     134
     13.2. Examination of the Crystallization Experiments     134
14. Seeding     139
Enrico A. Stura
     14.1. Streak Seeding     142
     14.2. Preparing Seed Stock     144
              Experiment 14.1. Streak seeding and seed transfer     144
     14.3. Diagnostic Uses of Seeding     145
     14.4. Seeding and Sitting Drops     146
     14.5. Optimization     146
     14.6. Seeding as a Screening Technique     147
     14.7. Heterogeneous Seeding     148
              Experiment 14.2. Epitaxial Jumps     149
     14.8. Recognizing an Epitaxial Jump     150
     14.9. Cross Seeding     150
     14.10. Summary     151
     A 14.1. Recommendations for Streak Seeding and Seed Transfer     153
15. Macroseeding: A Real-Life Success Story     155
Sherry L. Mowbray
     15.1. Why Do Macroseeding?     157
     15.2. How to Macroseed     158
              Step 1. Obtain seeds     158
              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     161
     15.3. Common Problems/Causes     161
              15.3.1. Too Many Crystals     161
              15.3.2. Dissolving Seeds     161
              15.3.3. Poor Crystals     161
16. Oils for Crystals     163
Naomi E. Chayen
     16.1. The Rationale for Crystallization under Oil     165
              16.1.1. The Microbatch Technique     166
              16.1.2. Setting up Crystallization Trials by Microbatch     166
              16.1.3. Crystallization of Membrane Proteins in Oil     168
              16.1.4. Additional Benefits of Oil     168
              16.1.5. Limitations of Crystallizing under Oil     168
              16.1.6. Harvesting Microbatch Crystals     169
              16.1.7. The Effect of Different Oils on Microbatch     169
     16.2. Use of Different Oils in Screening     169
     16.3. Use of Oils in Optimization     170
              16.3.1. Control of Nucleation     170
              16.3.2. Effect of Surface Contact on Nucleation     171
     16.4. The Use of Oil for Controlling the Rate of Vapor Diffusion Trials     172
     16.5. Summary     174
     Lab Exercises with Oils     175
            Exercise 16.1. Setting up microbatch trials     175
            Exercise 16.2. Filtration experiments     177
            Exercise 16.3. Containerless crystallization     178
            Exercise 16.4. Insertion of oil barrier to slow down vapor diffusion experiments     179
17. Crystallization for Cryo-Data Collection     181
Elspeth F. Garman
     17.1. Introduction     183
     17.2. Protocol for Finding Crystallization Conditions for Cryo-Crystallography     185
     17.3. Laboratory Exercise for Determining Cryo-Protectant Solutions     189
     17.4. Mounting Crystals for Room Temperature Data Collection     190
     17.4.1. General Procedure for Mounting a Crystal     190
     A 17.1. Glycerol Concentrations Required to Cryo-Protect CRYSTAL SCREEN I Solutions     194
18. Crystallization of Membrane Proteins     197
Jeff Abramson and So Iwata
     18.1. Principles of Membrane Protein Crystallization     199
     18.2. Practical Approach for Membrane Protein Crystallization     201
     18.3. Sample Preparation     202
              18.3.1. Selection of Detergent     202
              18.3.2. Purification     202
              18.3.3. Protein Concentration and Detergent Exchange     203
     18.4. Crystallization     203
              18.4.1. Selection of the Detergent     203
              18.4.2. Crystallization Setups     204
              18.4.3. Precipitants     207
              18.4.5. Temperature     207
              18.4.6. Screening Kits     207
     18.5. Optimization     207
19. One Last Piece of Advice: Appearances Can Be Deceiving!     211
Alex Cameron
A-Z Tips     215
Appendix A1. Good-to-Have Gizmos     251
     A1.1. Temperature Logger for the Crystallization Room     251
     A1.2. Positive Displacement Pipettes     251
     A1.3. Microelctrode for Measuring pH in the Drop Reservoir     252
     A1.4. Buffer Exchange for Small Volumes (<250 ml) of Proteins     253
     A1.5. Vortex Mixer Adapted for 24-Well Tissue Culture Plates     255
     A1.6. Automated Grease Dispenser     255
Appendix A2. Supplies for Crystallization and Suggested Sources     257
Appendix A3. Suppliers’ Addresses     261
Appendix A4. Useful Websites     269
Appendix A5. Commercially Available Screens     273

     A5.1. Crystal Screen I     273
     A5.2. Crystal Screen II     276
     A5.3. Additives Screens 1, 2, and 3     279
     A5.4. Wizard I     282
     A5.5. Wizard II     285
     A5.6. Cryo I     288
     A5.7. Cryo II     291
     A5.8. Structure Screen 1     294
     A5.9. Structure Screen 2     297
Index     301

Obtaining crystals is currently the bottleneck in protein structure determination by X-ray crystallography. The intent of this book is to collect the most current methods for crystallizing proteins and present them as lucid, easy-to-follow laboratory protocols. The accumulated knowledge on practical aspects of protein crystallization is spread out in many different sources or in the form of local lab lore. The value of a laboratory manual is that it organizes the practical portion of this knowledge. 
This book began from my own experiences in crystallizing proteins since l984, and from teaching my course “Practical Protein Crystallization”. The contributing authors of the book have included laboratory exercises where appropriate with their chapters so that the book can be used in such courses or to test the techniques before beginning on expensive, hard-won proteins. 
The first part of this book introduces the beginner to the basic techniques, materials, and parameters that affect crystallization. The more experienced student can turn directly to the chapters on dynamic light scattering and strategy. Dynamic light scattering is rapidly becoming an increasingly important diagnostic tool and an introductory, step-by-step guide is presented here. The protein, the star in this show, is sometimes purified by the crystallographer and sometimes by biochemists or molecular biologists. The protein purifier may not always know what handling considerations are important for a protein intended for crystallization experiments. Therefore I have included a chapter, based on questions from protein chemist collaborators through the years, to help identify the concerns in protein purification, which are unique for crystallization work.
Chapters 8 to 12 deal with crystallization strategy. It is essential to have a “map” or plan for searching the complicated multi-parametric space of crystallization conditions. If that plan does not work then change it, but by all means, begin with a plan of some sort. I have intentionally asked leading experts with conflicting approaches to present them. Thus, the contradictions that the reader finds among these chapters are not editorial oversights but a reflection of the current state of affairs in the screening problem.
Interpretation of results is probably the area in which beginners have the greatest difficulty. To my mind it is this ability to recognize the difference between a bad precipitate and a promising one, in the absence of any other leads, that constitutes the proverbial green thumb in crystallization. The pictorial guide in Chapter 13 helps you develop your own green thumb for growing protein crystals. 
The second part of the book takes up crystallization for cryo-crystallography, seeding, the use of oils, and crystallization of membrane proteins. As soon as the beginner has mastered the basic techniques, the contents of this part are essential for completing the “crystallization toolbox”. 
The A-Z section was the most fun part of this book to compile. Since I learned some new things myself in editing this section, I daresay it contains something of value for everyone, regardless of their level of experience. 
Some contents of the chapters overlap with material in other chapters. These repetitions have been permitted intentionally, with the reader in mind who will be dipping into the book, rather than reading it from cover to cover. 
The editor (that’s me) welcomes the readers to share their comments and experiences with the exercises and methods of this book by e-mail. ( 

I am indebted to the following companies and organizations for permission to use their figures or tables in this book: Hampton Research, Chemicon International Inc., Millipore Intertech, Emerald BioStructures, Molecular Dimensions, International Union of Crystallography (IUCr), Current Biology Ltd., and Pierce Chemical Co. 
I thank Drs. Matti Nikkola, Gerard Kleywegt and Professor Bror Strandberg for their critical reading of huge portions of this manuscript. My boss, Professor Alwyn Jones, allowed me to put aside the other interests of the lab for months so that I could work uninterruptedly on this book. Many colleagues in Uppsala contributed to this work and I thank Sherry Mowbray, Erling Wikman, Alex Cameron, Jill Sigrell, Mats Sandgren, Christina Divne, Inger Andersson, and Janos Hajdu for assistance and ideas. Stefan Knight, Brent Segelke, and Madeleine Riès-Kautt helped me with valuable discussions on crystallization 
The authors of the chapters generously took time from their own projects in order to create this book and it has been a privilege to work with them. Dr. Igor Tsigelny, at the publishers, International University Line, initiated the idea for this manual and saw it to its completion. I thank him for his support of this project. Stefan Odestedt and Carl Bergfors made this book possible by contributing many hours of baby-sitting and I am also indebted to S.O. for his help with the table of contents. Finally, I extend my heartfelt gratitude to my daughter, Carmen for her forbearance during my preoccupation with this book.

Uppsala Terese M. Bergfors
30 June 1998



Protein Crystallization: Techniques, Strategies, and Tips. A Laboratory Manual