Crystallization of Nucleic Acids and Proteins: A Practical Approach (Practical Approach Series) (2nd edition)

460 Pages·2000·23.64 MB·English

by  Arnaud Ducruix

Crystallization of Nucleic Acids and Proteins
The Practical Approach Series
SERIES EDITOR
B. D. HAMES
Department of Biochemistry and Molecular Biology
University of Leeds, Leeds LS2 9JT, UK
See also the Practical Approach web site at http://www.oup.co.uk/PAS
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Crystallization of
Nucleic Acids and
Proteins
A Practical Approach
Second Edition
Edited by
ARNAUD DUCRUIX
Laboratoire de Cristallographie et
RMN Biologiques, Faculte de Pharmacie
Universite de Paris V, Paris
and
RICHARD GIEGE
Institut de Biologie Moleculaire
et Cellulaire du CNRS, Strasbourg
OXFORD
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A catalogue record for this book is available from the British Library
Library of Congress Cataloging in Publication Data
Crystallization of nucleic acids and proteins : a practical approach /
edited by Arnaud Ducruix, Richard Giege — [2nd ed.]
(The practical approach series : 210)
Includes bibliographical references and index.
1. Proteins—Analysis. 2. Nucleic Acids—analysis. I. Ducruix.
A. (Arnaud) II. Giege, R. (Richard) III. Series.
QD431.25.A53C79 1999 547.7'5046—dc21 99-15222
ISBN 0-19-963679-6 (Hbk)
0-19-963678-8 (Pbk)
Typeset by Footnote Graphics,
Warminster, Wilts
Printed in Great Britain by Information Press, Ltd,
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Preface
With the development of genomics and proteomics, and their applications in
basic biological research and the biotechnologies, there is an increasing need
of three-dimensional structural knowledge of proteins, nucleic acids, and
multi-macromolecular assemblies by X-ray methods. To achieve this aim,
crystals diffracting at high resolution are needed. The major aim of the second
edition of this book in the Practical Approach series is to present an update of
the methods employed to produce crystals of biological macromolecules and
to outline the newest trends that have entered the field. Since the first edition,
which appeared in 1992, the science of crystallogenesis, which was then in its
infancy, has grown rapidly balancing between the physics of crystal growth and
blind-screen crystallizations. The advances can be appreciated from the Pro-
ceedings of the different International Conferences on the Crystallization of
Biological Macromolecules (ICCBM1 to 7) which appear every two years, the
latest covering ICCBM-7, held in June 1998 in Granada, and published in
J. Crystal Growth, Vol. 196, January 1999.
As usual in the series, the emphasis of the present book is to give detailed
laboratory protocols throughout the chapters. However, we have not given
protocols just as 'recipes', but instead we have intended to always present the
methods with reference to the theoretical concepts and principles underlying
them. In fact one of the aims of this book was to fight against the fallacious
idea according to which crystal growth of biological macromolecules is more
an 'art' than a science. Although this is probably sometimes true from a prac-
tical point of view, it is certainly incorrect in its principle. Therefore emphasis
has been given to the physical parameters involved in crystallization and on
the large knowledge on the crystal growth of small molecules, as well as to the
particular biochemical and physico-chemical properties of biological macro-
molecules.
This book is intended to be read by a wide range of scientists. First, by the
crystallographers who have to solve three-dimensional structures of macro-
molecules. Secondly, by all molecular biologists who have access to macro-
molecules but often do not know how to handle them for crystallization, and
who may consider crystallization as an esoteric undertaking, because of lack of
basic knowledge about the crystallization process. Thirdly, by the physico-
chemists and physicists who for other reasons consider biology as an esoteric
science. It is our wish that this book will contribute to a better understanding
of crystallogenesis by these scientists and to the improved perception of the
biological requirements that have to be taken into account for physical studies.
Finally, the book should be a laboratory guide for all students and beginners,
helping them to avoid making mistakes when entering the field of crystal
preparation.
Preface
Chapter 1 is an introduction to crystallogenesis of biological macro-
molecules. It includes a brief historical survey of the subject and introduces the
general principles and major achievements of this new discipline. The prepara-
tion of biological macromolecules and the concept of 'crystallography-grade
purity' are developed in Chapter 2. Chapter 3 is new and introduces the use of
molecular biology methods to 'customize' domains for structural biology. It
also includes the preparation of protein crystals made of protein molecules
containing selenomethionine residues and outlines why they can be used for
the multiple anomalous dispersion (MAD) method. Screen-like methods are
now widespread but do not provide suggestions when they fail. The answer
may then come from statistical methods presented in Chapter 4 which explains
their theory and gives practical advice (and a computer program) for protocol
design. One of the goals of this book is to give to crystal growers of biomacro-
molecules the conceptual and methodological tools needed to control crystal-
lization. This is examined in Chapter 5 which includes a description of the
classical crystallization methods together with workshop examples. Crystal-
lization in gels is described with theoretical and practical considerations in
Chapter 6. This chapter includes a novel section describing the gel acupunc-
ture method; it also contains information on crystal growth under microgravity
and hypergravity conditions. Because it is sometimes difficult to reproduce
appropriate nucleation conditions, Chapter 7 is devoted to seeding procedures
with preformed crystalline material, including micro-, macro-, and cross-
seeding with numerous examples.
The special cases of nucleic acids (and their complexes with proteins and
nucleoprotein assemblies) and membrane proteins are covered in two indi-
vidual chapters (8 and 9). Their crystallization is still challenging, but the
novel developments in the field have led to a number of recent breakthroughs,
that are encouraging for experimenters entering the field. For nucleic acids,
Chapter 8 gives emphasis to the strategies for the design and preparation of
appropriate DNA or RNA fragments and to the specific features character-
izing their crystallization, either free or in complexes with proteins. For
membrane proteins, the already published genomes show that a good third of
the expressed proteins belong to this category and it is expected that the inter-
est for membrane proteins will expand quickly, especially among structural
biologists. It is our wish that the methods described in Chapter 9 will help them
to reach this goal.
The link between protein solubility and the physico-chemical parameters
governing crystal growth is presented in Chapter 10 with a strong emphasis on
the practical issues. Chapter 11 deals with physical methods and gives an intro-
duction to the physics of crystal growth. In particular, the use of light scattering
methods to monitor early nucleation events is advocated with examples and a
description of the material used.
Chapter 12 is new and covers the expanding field of the two-dimensional
crystallization of soluble proteins on planar lipid films. It presents many proto-
Preface
cols which may be readily used. Soaking of crystals of biological macro-
molecules is of great interest for crystallographers, either for resolving a
structure (heavy-atom derivatives), or for diffusing inhibitors, activators, or
cofactors (eventually photoactivable). As in all previous chapters, practical
aspects were the driving force and Chapter 13 is illustrated by a variety of pro-
tocols. The editors thought that an introduction to X-ray crystallography
should be included in this book. This is done in Chapter 14, that is geared
toward biochemists wanting to characterize crystals by themselves rather than
explaining how to solve a structure.
It is a great pleasure to acknowledge our gratitude to a number of friends
and colleagues. First, our colleagues from Gif-sur-Yvette/Paris and Strasbourg
deserve particular thanks for having participated over the years in the develop-
ment of our studies on crystallogenesis; their enthusiasm was essential and
gave us the impetus for the preparation of a book covering this field and for
updating it in its second edition. However, without the invaluable help of
many friends from both sides of the Atlantic who agreed to cover specialized
topics, this venture would not have been possible. We would like to warmly
thank all of them. The French Centre National de la Recherche Scientifique
(CNRS) and Centre National d'Etudes Spatiales (CNES) are acknowledged
for their permanent support in developing biological crystallogenesis and their
interest for the physico-chemical aspects of the field.
While preparing this second edition, our dear colleague Roland Boistelle
closed his eyes. He was a source of inspiration for all of us and one of the first
who geared the biology oriented scientists to the physics of crystal growth. His
contribution to the field of macromolecules crystallogenesis was essential and
we would like to dedicate this book to his memory.
Paris and Strasbourg A. D.
June 1999 R. G.
IX