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Evolving Methods for Macromolecular
Crystallography
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NATO Science Series
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Series II:Mathematics,Physics and Chemistry – Vol.245
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Evolving Methods for
Macromolecular Crystallography
The Structural Path to the
Understanding of the Mechanism
of Action of CBRN Agents
Edited by
Randy J. Read
Department of Haematology, University of Cambridge, Cambridge Institute
for Medical Research, Cambridge, U.K.
Joel L. Sussman
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
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Proceedings of the NATO Advanced Study Institute on
Evolving Methods for Macromolecular Gystallography:
The Structural Path to the Understanding of the Mechanism
of Action of CBRN agents
Erice, Italy
19—28 May 2005
AC.I.P. Catalogue record for this book is available from the Library of Congress.
ISBN 978-1-4020-6315-2 (PB)
ISBN 978-1-4020-6314-5 (HB)
ISBN 978-1-4020-6316-9 (e-book)
Published by Springer,
P.O. Box 17, 3300 AADordrecht, The Netherlands.
www.springer.com
Printed on acid-free paper
All Rights Reserved
©2007 Springer
No part of this work may be reproduced, stored in a retrieval system, or transmitted in any
formor by any means, electronic, mechanical, photocopying, microfilming, recording or
otherwise, without written permission from the Publisher, with the exception of any material
supplied specifically for the purpose of being entered and executed on a computer system, for
exclusive use by the purchaser of the work.
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TABLE OF CONTENTS
PREFACE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
1. SUCCEEDING WITH SEEDING:
SOME PRACTICAL ADVICE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Terese Bergfors
2. EXPRESSION, PURIFICATION, AND CRYSTALLISATION
OF MEMBRANE PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Bernadette Byrne
3. MACROMOLECULAR CRYO-CRYSTALLOGRAPHY. . . . . . . . . 25
Elspeth Garman
4. PROCESSING DIFFRACTION
DATA WITH MOSFLM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Andrew G.W. Leslie and Harold R. Powell
5. SAD PHASING: BASIC CONCEPTS AND
HIGH-THROUGHPUT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
George M. Sheldrick
6. LIKELIHOOD-BASED EXPERIMENTAL
PHASING IN PHASER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Airlie J. McCoy, Laurent C. Storoni, and Randy J. Read
7. STOCHASTIC MOLECULAR REPLACEMENT. . . . . . . . . . . . . . 79
Nicholas M. Glykos
8. LIKELIHOOD-BASED MOLECULAR
REPLACEMENT IN PHASER. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Randy J. Read, Airlie J. McCoy, and Laurent C. Storoni
9. AUTOMATED STRUCTURE DETERMINATION
WITH PHENIX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Paul D. Adams, Pavel V. Afonine, Ralf W. Grosse-Kunstleve, Nigel
W.Moriarty,Nicholas K.Sauter,Peter H.Zwart,Kreshna Gopal,
Thomas R.Ioerger,Lalji Kanbi,Erik McKee,Reetal K.Pai,
Li-Wei Hung,Thiru Radhakannan,Airlie J.McCoy,
Randy J.Read,Laurent C.Storoni,Tod D.Romo,
James C.Sacchettini,and Thomas C.Terwilliger
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vi TABLE OF CONTENTS
10. DENSITY MODIFICATION IN MAIN . . . . . . . . . . . . . . . . . . . . 111
Dusˇan Turk
11. AB INITIO PHASING STARTING FROM
LOW RESOLUTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Vladimir Lunin, Natalia Lunina,
and Alexandre Urzhumtsev
12. STRUCTURAL GENOMICS OF MYCOBACTERIUM
TUBERCULOSIS: A SEARCH FOR FUNCTION
AND NEW DRUG TARGETS. . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Ted Baker
13. THREE-DIMENSIONAL DOMAIN SWAPPING AND
ITS RELEVANCE TO CONFORMATIONAL
DISEASES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Mariusz Jaskolski
14. STRUCTURAL BIOINFORMATICS: FROM PROTEIN
STRUCTURE TO FUNCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . 165
James D. Watson, Adel Golovin, Roman A. Laskowski,
Kim Henrick, Janet M. Thornton, Andrzej Joachimiak,
and Aled M. Edwards
15. SINGLE-PARTICLE IMAGING. . . . . . . . . . . . . . . . . . . . . . . . . . 181
David Sayre
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PREFACE
This volume comprises papers presented at the 2005 edition of the
“Crystallography of Molecular Biology” courses that have been held since
1976 at the Ettore Majorana Centre for Scientific Culture in Erice,Italy.This
series of courses is renowned for bringing leaders in the field of macromole-
cular crystallography together with highly motivated students,in a beautiful
and intimate location that encourages people to interact. The warm and
informal atmosphere at these Erice conferences, especially these on crystal-
lography,has helped to foster long-term scientific interactions and new inter-
national friendships that have often lasted for the lifetime of the scientists.
The course was financed by NATO as an Advanced Study Institute and by
the European Commission as a EuroSummerSchool.
The papers span the breadth of material presented in the course, which
emphasized the practical aspects of modern macromolecular crystallography
and its applications. One must start with crystals: Bergfors showed how to
improve initial crystals through seeding, while Byrne discussed the difficult
problem of crystallizing membrane proteins. The collection of optimal
diffraction data requires both careful preparation of cryo-cooled crystals
(Garman) and proper processing of the diffraction images (Leslie). To
obtain images of electron density, one needs estimates of the phases of the
diffracted spots. Sheldrick presented the background to the single-
wavelength anomalous diffraction (SAD) method, which has been gaining
popularity, and McCoy discussed the basis of modern maximum likelihood
methods for treating information in experimental phasing. When a related
structure is known, the phases can be obtained by molecular replacement,
which can use stochastic search methods (Glykos) or tree search methods
based on maximum likelihood (Read). There is also the promise that ab
initio phasing methods will contribute at least at low resolution (Lunin).
Initial phases can be improved dramatically by density modification (Turk).
Increasingly, all these methods can be automated (Adams), an important
step to increasing the throughput of structural genomics efforts (Baker). At
times,structural genomics provides structures without a known function,but
Thornton showed that structure alone can shed light on function. Careful
analysis of structures can provide an explanation for disease processes at the
atomic level (Jaskolski). The climax of this volume, as of the course, is
the demonstration by Sayre that diffraction can be used to image single
particles as large as cells.
Most of the real organizational work for the course was done by Paola
Spadon and Lodovico Riva di Sanseverino,who,between them,found most
of the funding, corresponded with applicants and selected participants,
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viii PREFACE
made the logistical arrangements,and reminded us patiently when we needed
to do something. Lodovico brought a wealth of experience to bear, having
been a mainstay of the Erice meetings since their inception. John Irwin
played an essential role,organizing all the information technology (IT) facil-
ities needed to conduct tutorials and demonstrations in a computer (CPU)-
intensive field like macromolecular crystallography.
Paola, Lodovico, and John were joined as Fellows of the Loyal Order of
Orange Scarves by a set of enthusiastic volunteers: Vito Calderone, Laura
Cendron, Sonia Covaceuszach, Federica Morandi, Elena Papinutto, Nicola
Pasquato,Fabiana Renzi,and Donatella Tondi.Together they dealt with any
of the day-to-day emergencies that arise in running a course like this.
In addition to the essential support from NATO and the European
Commission,generous financial support was received from the International
Union of Biochemistry and Molecular Biology, INTAS, the International
Union of Crystallography, the University of Bologna, AstraZeneca, CCP4,
and Douglas Instruments.
Randy J. Read and Joel L. Sussman
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SUCCEEDING WITH SEEDING: SOME PRACTICAL ADVICE
TERESE BERGFORS
Department of Cell and Molecular Biology,Uppsala University
Biomedical Center,Box 596,751 24 Uppsala,Sweden
Abstract: Seeding is a powerful and versatile method for optimizing crystal
growth conditions.This article discusses,from a practical point of view,what
seeding is,the selection and transfer of seeds,and into what conditions they
should be transferred.The most common causes offailures in seeding experiments
are also analyzed.
Keywords:crystallization;microseeding;optimization;seeding;streak seeding.
1. Introduction
Crystallization is the rate-limiting step in the process of determining a
three-dimensional macromolecular structure by X-ray crystallography.
Automation and miniaturization of the crystallization setup have greatly
facilitated massive screening. However, screening in itself, no matter how
extensive, is still no guarantee that crystals will be found or that they will
provide diffraction-quality data.At least half of the crystals obtained in an initial
screen cannot be used without further optimization [1]. Therefore, optimization
methods are often crucial for the success of a crystallization project.
One powerful tool in the arsenal of optimization techniques is seeding.
While it is not a universal solution to all optimization problems, seeding is
relatively cheap,fast,and easy,which makes it worth trying at an early stage.
Possible applications include:
● If spontaneous nucleation is slow,i.e.,the drop stays clear for a long time
(weeks to months) before crystals appear
● To reduce showers of crystals
● To increase the size of crystals
● To improve reproducibility due to erratic nucleation, i.e., supposedly
identical drops do not consistently produce crystals
● If crystals grow in clusters rather than singly
1
R.J.Read and J.L.Sussman (eds.):Evolving Methods for Macromolecular Crystallography,1–10
©2007.Springer
