Table Of Content• • • • •
PRACTICAL
PROTEIN
CRYSTALLOGRAPHY
• • • • •
DUNCAN E. McREE
Department of Molecular Biology
The Scripps Research Institute
Lajolla, California
ACADEMIC PRESS, INC.
A Division of Harcourt Brace & Company
San Diego New York Boston London Sydney Tokyo Toronto
Cover ilustration: Electron density map (2Fo-Fc, 2.0 A) of endonuclease III superimposed
on the model of [4Fe-4S] cluster and surrounding protein. The map has been colored
according to the gradient of the density in increasing order, (green, red, yellow, blue) in
order to color carbon density green, oxygens red, sulfurs yellow, and irons blue. This
picture was made using XtalView and AVS (Advanced Visual Systems) software by Mike
Pique, Maria Thayer, and John Tainer of The Scripps Research Institute.
This book is printed on acid-free paper. @>
Copyright © 1993 by ACADEMIC PRESS, INC.
All Rights Reserved.
No part of this publication may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopy, recording, or any information
storage and retrieval system, without permission in writing from the publisher.
Academic Press, Inc.
1250 Sixth Avenue, San Diego, California 92101-4311
United Kingdom Edition published by
Academic Press Limited
24-28 Oval Road, London NW1 7DX
Library of Congress Cataloging-in-Publication Data
McRee, Duncan Everett, Date.
Practical protein crystallography / Duncan E. McRee
p. cm.
Includes bibliographical references and index.
ISBN 0-12-486050-8
1. Proteins—Structure. 2. Crystallography. I. Title.
QP551.M366 1993
574. 19' 245-dc20 93-13272
CIP
PRINTED IN THE UNITED STATES OF AMERICA
93 94 95 96 97 98 BB 9 8 7 6 5 4 3 2 1
• • • • • • • • • • • • • • ••
PREFACE
• • • • • • • • • • • • • • ••
This book is a practical handbook intended to be used by anyone who
wants to solve a structure by protein crystallography. It should prove useful
both to new protein crystallographers and to old hands. While users of this
handbook should be familiar with at least one of the texts in the Suggested
Reading list that follows, Practical Protein Crystallography examines several
subjects that are not covered in these other texts, but which are widely used
today, for example, area detectors and synchrotron radiation sources. The
topics covered in this book are well-tested, robust methods commonly used
in our laboratory and in others. The informed crystallographer will note,
however, that many techniques and methods are not mentioned or are men
tioned very briefly. These have been omitted due to space, less common use,
difficulty of application, the need for special expertise, or perhaps oversight
on the author's part. The exclusion of a method should not be taken in any
way to be a disapproval; it is simply not possible for one book to include
everything.
The book is divided into four chapters: (1) Laboratory Techniques, (2)
Data Collection Techniques, (3) Computational Techniques, and (4) Protein
Crystallography Cookbook. Techniques and methods to be practiced at the
lab bench are in Chapter 1, methods used for collection of diffraction data are
in Chapter 2, and those done at the computer keyboard are in Chapter 3.
Chapter 4 contains examples of projects to provide "recipes" for solving a
project. It presents a means for tying together several different methods and
includes real numbers to give a better understanding of results. There is no
need to read the book in any order; chapters that use information from other
xi
xii PREFACE
chapters will reference those chapters when necessary. In fact, the best use of
the book may be to read the Cookbook first and then refer to other chapters
as needed.
In Chapter 1, only cursory information on crystal growth is presented
because several excellent texts on crystal growth already exist. A fair amount
of attention is given to protein sample handling, because proper attention to
the sample can often mean the difference between a successful project and a
failure. Proteins are delicate materials that demand special handling and are
very difficult to purify in large quantity. Protein crystals are also very delicate
and require special handling techniques different from those of small mole
cule crystals.
Chapter 2 bridges the gap between the laboratory and the computer.
Special emphasis is placed on the newer techniques using area detectors and
synchrotron sources that, except for special cases, will soon replace the older
film and single counter diffractometer methods, which are covered only
briefly. Because of the high cost of these systems, the user will probably have
access to only one. Experience has shown that the user will then come to
regard this one as best and will defend it vehemently against all others. Em
phasis is placed less on a specific system and more on general techniques rele
vant to all area detectors.
Chapter 3 openly acknowledges the integral role that computers play in
modern crystallography. Not so many years ago, protein crystallographic
software was stored on punched cards and data was stored on paper tape.
Now, most of us can't find a working card reader. While the hardware used
by protein crystallography has surged ahead, much of the software being used
was originally intended for punch cards. Hopefully, the reader will use the
information in this book as a guide into the modern age of computing. The
variety of software used by different groups is enormous and no book could
hope to cover even a small portion of it. General information that should be
applicable to most techniques is given. Although this book can never substi
tute for the individual manuals of each program, it does give guidelines that
will allow the reader to make intelligent choices among the program options.
In order to allow discussion of specifics, the XtalView system is used. This
system employs a visual interface that is easy to grasp and illustrates well the
possible options at a given step. XtalView is available from the Computational
Center for Macromolecular Structure, San Diego Supercomputer Center, P.O.
Box 85608, San Diego, CA 92186-9784, phone (619) 534-5100. Send e-mail
to [email protected] requesting more information on XtalView. If
you do not have access to e-mail then write to the above address.
Chapter 4 contains examples drawn from the experience of the author
and his colleagues, which provides some examples of protein structures solved
by various methods. These examples can be used as guides for the user's own
PREFACE xiii
projects and to give a feel for how to apply the varied methods. Real numbers
are given as a basis for interpreting the user's own. By following the examples
in multiple isomorphous replacement users can, with luck and perseverance,
solve their own structure.
Appendix A contains formulae commonly used in protein crystallogra
phy, but with a twist: the formulae are coded in both FORTRAN77 and C.
These two languages easily account for 99% of all protein crystallographic
software. This will be of great aid to users in writing their own software and
in understanding other software. Also for those of us who understand a com
puter language better than we do math, this appendix explains the formulae.
One goal of this book is to provide enough information so that the computer
neophyte can write a simple program to reformat or filter data. Unfortunately,
because of the incredible variety of software available and the consequent
large variety of file formats, this is a necessary skill. Appendix A provides
information to help write programs that will continue to be useful on different
operating systems and for other projects. It is surprising how many programs
that were originally written to be used once for a single problem are now
commonly used by a dozen laboratories — all wishing the original writer had
made the program more general.
Appendix Β contains the manual of the XtalView system. Much practi
cal information is contained here that will answer specific questions about file
formats and options available. Again, this is only one of many systems avail
able, but it will serve to give a practical handle on the nature of crystallo
graphic computing.
Rules of thumb are provided throughout to serve as a guide. Like all
rules, these are made to be broken and should not be taken too literally. Every
protein has its vagaries and its own special difficulties —otherwise several
thousand protein structures would have been solved to date instead of a few
hundred. The low ratio of proteins with published coordinates to the number
of proteins reported as crystallized makes it plain that many protein struc
tures take years to be solved and that many will never be solved. Perseverance
and a willingness to try new methods have been keys to solving many struc
tures. Protein crystallography is not a turn-key science. But that is part of its
fascination and challenge.
Suggested Reading
Crystallography
Stout, G. H., and Jensen, L. H. (1989). X-Ray Structure Determination: A Practical
Guide, 2nd Ed. Wiley, New York.
Ladd, M. S. B., and Palmer, R. A. (1985). Structure Determination by X-Ray Crystallog
raphy, 2nd Ed. Plenum, New York.
xiv PREFACE
McKie, D., and McKie, C. (1986). Essentials of Crystallography. Blackwell Scientific,
Oxford.
Protein Crystallography
Blundell, T. L., and Johnson, L. N. (1976). Protein Crystallography. Academic Press, San
Diego.
Wyckoff, H., ed. (1985). In Diffraction Methods for Biological Macromolecules, Methods
in Enzymology, Vols. 114 and 115. Academic Press, San Diego.
Helliwell, J. R. (1992). Macromolecular Crystallography with Synchrotron Radiation.
Cambridge Univ. Press, Cambridge.
Crystallization
McPherson, Α., Jr. (1982). The Preparation and Analysis of Protein Crystals. Wiley, New
York.
Protein Structure
Dickerson, R. E., and Geis, I. (1969). The Structure and Action of Proteins, Benjamin/
Cummings Publishing Co., Menlo Park, California.
Fersht, A. (1985). Enzyme Structure and Mechanism. W. H. Freeman, New York.
Oxender, D. L., and Fox, C. F., eds. (1987). Protein Engineering. Alan Liss, New York.
• • • • • • • • • • • • • • ••
ACKNOWLEDGMENTS
• • • • • • • • • • • • • • •Φ
My thanks go to a number of people for their assistance in preparing
the manuscript of this book. In particular I thank David Stout, Yolaine Stout,
and Michele McTigue for critically reading the manuscript and making many
helpful suggestions. Any errors in the book are, of course, my responsibility.
I thank John Tainer, Elizabeth Getzoff, Hans Parge, Susan Redford, Gloria
Borgstahl, Brian Crane, David Goodin, Ch-fu Kuo, Zhong Ren, Andrew Ar-
vai, Peter Lauble, Art Robbins, and David Stout for allowing me to use their
work as examples in this book. I also thank the large number of patient people
in the structural biology group at The Scripps Research Institute who beta-
tested XtalView for me. I hope that this book answers a few of their questions
about what to do with all those buttons and sliders. The Scripps Research
Institute has provided me with an outstanding environment and the freedom
for learning and practicing protein crystallography. I especially thank Mi
chael Pique who patiently taught me computer graphics over the years and
who, along with Lynn Ten Eyck and Andrew Arvai, provided me with rou
tines for XtalView.
I have worked with many people over the years who have provided the
intellectual stimulus and help that made this book possible. David Richard
son was my Ph.D. advisor and started me out on the right foot. Jane Richard
son has been a major inspiration over the years. Wayne Hendrickson took me
under his wing and taught me much about anomolous scattering, phasing,
refinement, scaling, and critical thinking. Bi-Cheng Wang and Bill Furey
taught me all about phase modification in a couple of visits to the University
of Pittsburgh. Fred Brooks made me think a lot about the virtues of a good
XV
xvi ACKNOWLEDGMENTS
user interface and the real power of computer graphics. John Tainer and Eliz
abeth Getzoff have been invaluable as partners in solving an ever-increasing
number of interesting protein structures.
XtalView distribution through the Center for Macromolecular Struc
ture at the San Diego Supercomputer Center is funded by Grant DIR 8822385
from the National Science Foundation.
Finally, I thank my wife, Janice Yuwiler, and her father, Art Yuwiler, for
always believing that the book would one day be finished. And last, but not
least, I want to thank my sons Alex and Kevin for putting up with me while I
spent so many evenings late at work.
1
• • • • JL • • • •
LABORATORY
TECHNIQUES
• • • • • • • • • ••
••••• 1·1 •••••
PREPARING PROTEIN SAMPLES
A protein sample must be properly prepared before it can be used in a
crystal-growth experiment. There are many ways that this can be done to
accomplish the same goal: to put the protein at a high concentration in a
defined buffer solution. Methods that have worked well in our laboratory are
outlined here, but if you know of a quicker, easier method then by all means
use it. The steps needed to prepare a protein sample are detailed below.
History and Purification
Since it is not uncommon for one batch of protein to crystallize while
the next will not, it is vital to keep a history of each sample and to track each
batch separately. Your records may provide the only clue as to the differences
between samples that produce good crystals and samples that are unusable.
For example, there have been several cases where the presence of a trace
metal is needed for crystallization. The most famous case is insulin. It seemed
that the only insulin that would crystallize was that purified from material
collected in a galvanized bucket. It was eventually discovered that zinc was
required, and later it was added directly to the crystallization mix. In our lab
1
2 LABORATORY TECHNIQUES
any sample received is logged into a notebook with a copy of any letters or
material sent with the sample.
Samples should be shipped to you on dry ice and kept frozen at - 70° C
until they are ready for use. Ask the person sending the sample to aliquot the
protein into several tubes and to quick-freeze each one. This way you can
thaw one aliquot at a time without having to repeatedly freeze-thaw the
entire sample, which can damage many proteins. Keep a small portion of the
sample apart and save it for future comparison with samples that do not
crystallize or crystallize differently.
Always keep protein samples at 4° C in an ice bucket to prevent dena-
turation and to retard bacterial growth. Perform all sample manipulations at
4° C either in a cold room or on ice. When the samples are finally set up they
can be brought to room temperature. Proteins are usually stabilized by the
presence of the precipitants used in crystallization and agents are added to
retard microbial and fungal growth. A common anti-microbial agent is
0.02% sodium azide. Other broad-spectrum cocktails are sold for use with
tissue culture that are quite effective.
Exchanging Buffers
If the desired buffer of the sample is not already known, the sample
should be placed in a weak buffer near neutrality. A good choice is 50 mM
Tris-HCl at pH 7.5, 0.02% sodium azide. Some proteins will not be stable
at low ionic strength, so a small portion should be tried first to see if a pre
cipitate forms. Be sure to wait several hours before deciding if the sample is
stable. Observe the sample in a clear glass vial and hold it near a bright light
to detect any cloudiness in the sample.
There are two methods for exchanging the buffer solution of the pro
tein sample: dialysis and use of a desalting column. The desalting column is
the fastest method, and if disposable desalting columns such as a PD-10 col
umn from Pharmacia-LKB is used, it is very convenient. A single pass
through the column will remove 85-90% of the original salt in the sample
and, if this is not enough, two passes can be used. Every time the sample is
passed over the column it will be diluted about 50%. Unless the sample is
very concentrated to begin with, it may be necessary to concentrate the
sample after desalting.
Dialysis on small volumes is best carried out in finger-shaped dialysis
membrane such as a colloidicon (Fig. 1.1). Always soak the membrane first
in the buffer to remove the storage solution. A minimum of two changes of
dialysis buffer 12 h apart is recommended. The dialysis buffer should be
stirred and should be at least 100 times the sample volume. Use membranes
with a molecular weight cutoff less than half of the sample molecular weight,
