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. (email@example.com).
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