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Structural Proteomics
 Methods in Molecular BiologyTM
John M. Walker, Series Editor
450. Germline Stem Cells, edited by Steven X. Hou and Shree Ram Singh, 2008.
449. Mesenchymal Stem Cells: Methods and Protocols, edited by Darwin J. Prockop, Douglas G. Phinney, and
Bruce A. Brunnell, 2008.
448. Pharmacogenomics in Drug Discovery and Development, edited by Qing Yan, 2008.
447. Alcohol: Methods and Protocols, edited by Laura E. Nagy, 2008
446. Post-translational Modification of Proteins: Tools for Functional Proteomics, Second Edition,
edited by Christoph Kannicht, 2008
445. Autophagosome and Phagosome, edited by Vojo Deretic, 2008.
444. Prenatal Diagnosis, edited by Sinhue Hahn and Laird G. Jackson, 2008.
443. Molecular Modeling of Proteins, edited by Andreas Kukol, 2008
442. RNAi: Design and Application, edited by Sailen Barik, 2008
441. Tissue Proteomics: Pathways, Biomarkers, and Drug Discovery, edited by Brain Liu, 2008
440. Exocytosis and Endocytosis, edited by Andrei I. Ivanov, 2008
439. Genomics Protocols: Second Edition, edited by Mike Starkey and Ramnanth Elaswarapu, 2008
438. Neural Stem Cells: Methods and Protocols, Second Edition, edited by Leslie P. Weiner, 2008
437. Drug Delivery Systems, edited by Kewal K. Jain, 2008
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435. Chromosomal Mutagenesis, edited by Greg Davis and Kevin J. Kayser, 2008
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433. Gene Therapy Protocols: Volume 1: Production and In Vivo Applications of Gene Transfer Vectors, edited
by Joseph M. LeDoux, 2008
432. Organelle Proteomics, edited by Delphine Pflieger and Jean Rossier, 2008
431. Bacterial Pathogenesis: Methods and Protocols, edited by Frank DeLeo and Michael Otto, 2008
430. Hematopoietic Stem Cell Protocols, edited by Kevin D. Bunting, 2008
429. Molecular Beacons: Signalling Nucleic Acid Probes, Methods and Protocols, edited by Andreas Marx
and Oliver Seitz, 2008
428. Clinical Proteomics: Methods and Protocols, edited by Antonio Vlahou, 2008
427. Plant Embryogenesis, edited by Maria Fernanda Suarez and Peter Bozhkov, 2008
426. Structural Proteomics: High-Throughput Methods, edited by Bostjan Kobe, Mitchell Guss, and Thomas
Huber, 2008
425. 2D PAGE: Volume 2: Applications and Protocols, edited by Anton Posch, 2008
424. 2D PAGE: Volume 1: Sample Preparation and Pre-Fractionation, edited by Anton Posch, 2008
423. Electroporation Protocols, edited by Shulin Li, 2008
422. Phylogenomics, edited by William J. Murphy, 2008
421. Affinity Chromatography: Methods and Protocols, Second Edition, edited by Michael Zachariou, 2008
420. Drosophila: Methods and Protocols, edited by Christian Dahmann, 2008
419. Post-Transcriptional Gene Regulation, edited by Jeffrey Wilusz, 2008
418. Avidin–Biotin Interactions: Methods and
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415. Innate Immunity, edited by Jonathan Ewbank and Eric Vivier, 2007
414. Apoptosis in Cancer: Methods and Protocols, edited by Gil Mor and Ayesha Alvero, 2008
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412. Neutrophil Methods and Protocols, edited by Mark T. Quinn, Frank R. DeLeo, and Gary M. Bokoch, 2007
411. Reporter Genes: A Practical Guide, edited by Don Anson, 2007
Structural Proteomics
High-Throughput Methods

Edited by

Bostjan Kobe
School of Molecular and Microbial Sciences, The University of Queensland, Brisbane,
Queensland, Australia

Mitchell Guss
School of Molecular and Microbial Biosciences, University of Sydney, Sydney,
Australia

Thomas Huber
School of Molecular and Microbial Sciences and Australian Institute for Bioengineering
and Nanotechnology, The University of Queensland, Brisbane, Australia
Bostjan Kobe Mitchell Guss
School of Molecular and Microbial Sciences School of Molecular and Microbial Biosciences
The University of Queensland University of Sydney, Sydney
Brisbane, Queensland Australia
Australia

Thomas Huber
School of Molecular and Microbial Sciences
and Australian Institute for Bioengineering
and Nanotechnology
The University of Queensland, Brisbane
Australia

ISBN: 978-1-58829-809-6 e-ISBN: 978-1-60327-058-8

Library of Congress Control Number: 2007937390

© 2008 Humana Press, a part of Springer Science+Business Media, LLC

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher
(Humana Press, 999 Riverview Drive, Suite 208, Totowa, NJ 07512 USA), except for brief excerpts in connection with reviews
or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer
software, or by similar or dissimilar methodology now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such,
is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors
nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher
makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

9 8 7 6 5 4 3 2 1

springer.com
 Preface

Structural genomics is a newly emerging field that has arisen following the
successful footsteps of the major sequencing efforts generally bundled under
the heading genomics. Practical considerations and the diversity of funding
mechanisms in different countries have led to different interpretations of what
structural genomics actually is. By a strict analogy to sequencing, one might envisage
structural genomics to be the determination of the three-dimensional structures
of all the proteins coded by a genome. This is an impractical goal because many
proteins are not amenable to purification in a form suitable for structure determi-
nation, or in fact may be inherently unstructured. Furthermore, the numbers of
distinct polypeptides produced in a eukaryotic organism—when one takes into
account the possibility of splice variants and posttranslational modification—is so
large that determining the structures of all forms would be an impossible task at
the present time.
The National Institute of Health (NIH) in the United States defined the
task of their structural genomics effort as the determination of a representa-
tive of every possible protein fold. Estimates of the total number of different
folds vary from a few as 1,000 to nearly 10,000; therefore, the structures in
these projects would not come from a single genome. Having at least one
representative of each fold would enable related structures in other organisms
to be modeled by homology and therefore provide the basis for modeling the
structure for every protein in an organism even if the structures could not be
determined experimentally. Substantial progress has been made toward this
goal. The success of this strategy for determining new folds is evidenced by
new submissions to the Protein Data Bank, in which more than 50% of struc-
tures submitted in 2006 with folds unrelated to those already deposited in the
Protein Data Bank were determined by structural genomics consortia.
Structural genomics in Europe, Japan, and elsewhere have generally had a
slightly different focus than the NIH-funded initiatives. These have targeted
pathogenic organisms or potential targets for drug intervention. Other smaller-
scale structural genomics projects might more properly be termed “structural
proteomics,” in which proteins are chosen based on their involvement in a
particular disease state, biochemical pathway, or environmental niche.
Whatever their overall aim, structural genomics consortia throughout the
world have made a major contribution to the development of new techniques

v
vi Preface

and methods for all aspects of structural biology, ranging from the identi-
fication of target genes to the refinement of structures using either nuclear
magnetic resonance or x-ray crystallographic techniques. Given that structural
genomics of whatever flavor deals with large numbers of structures, the new
and optimized methods enable high throughput processing of large numbers
of samples. It may fairly be said that high throughput is really the defining
characteristic of structural genomics.
The objective of this volume of Methods in Molecular Biology, Structural
Proteomics: High-Throughput Methods, is to provide readers with a current
view of all aspects of the “pipeline” that takes protein targets to structures and
how these have been optimized. Given the wide variety of approaches taken in
different laboratories, some individual methods will no doubt have been omit-
ted despite a genuine attempt on the part of the editors and authors to cover
their topic areas as widely as possible. This volume includes chapters describ-
ing the individual steps in the structural genomics pipeline in depth, as well as
less detailed overviews of individual structural genomics initiatives. The over-
views give some insight into the diversity of approaches adopted by different
laboratories. The chapters are grouped in sections ordered in progression along
the structural genomics pipeline: “Protein Target Selection, Bioinformatic
Approaches and Data Management,” “Protein Production,” “Biophysical
and Functional Characterization of Proteins,” “Structural Characterization
of Proteins,” and “Structural Proteomics Initiatives Overviews.” Readers are
encouraged to access further details on the methodologies in online resources
such as PepcDB (protein expression purification and crystallization database:
pepcdb.pdb.org) and the cited literature. It should be emphasized that most
methods are as amenable to small laboratories as large consortia, and do not
require major investments in facilities. It is hoped that this volume will help
smaller laboratories establish high-throughput techniques.
Bostjan Kobe, BSc, PhD
Mitchell Guss, BSc, PhD
Thomas Huber, PhD
 Contents

Preface........................................................................................................ v

Contributors ............................................................................................... xiii

Section I. Protein Target Selection, Bioinformatic Approaches,

and Data Management

1 Target Selection for Structural Genomics: An Overview ................. 3

Russell L. Marsden and Christine A. Orengo

2 A General Target Selection Method

for Crystallographic Proteomics ....................................................... 27
Gautier Robin, Nathan P. Cowieson, Gregor Guncar,
Jade K. Forwood, Pawel Listwan, David A. Hume,
Bostjan Kobe, Jennifer L. Martin, and Thomas Huber

3 Target Selection: Triage in the Structural Genomics Battlefield ...... 37

James Raftery

4 Data Management in Structural Genomics: An Overview ............... 49

Sabrina Haquin, Eric Oeuillet, Anne Pajon, Mark Harris,
Alwyn T. Jones, Herman van Tilbeurgh, John L. Markley,
Zolt Zolnai, and Anne Poupon

5 Data Deposition and Annotation at the Worldwide

Protein Data Bank ............................................................................. 81
Shuchismita Dutta, Kyle Burkhardt, Ganesh J. Swaminathan,
Takashi Kosada, Kim Henrick, Haruki Nakamura,
and Helen M. Berman

6 Prediction of Protein Disorder .......................................................... 103

Zsuzsanna Dosztányi and Peter Tompa

vii
viii Contents

7 Protein Domain Prediction ............................................................... 117

Helgi Ingolfsson and Golan Yona

8 Protein Structure Modeling with MODELLER ............................... 145

Narayanan Eswar, David Eramian, Ben Webb, Min-Yi Shen,
and Andrej Sali

Section II. Protein Production

9 High Throughput Cloning with Restriction Enzymes ...................... 163

Volker Sievert, Asgar Ergin, and Konrad Büssow

10 Automated Recombinant Protein Expression Screening

in Escherichia coli ............................................................................ 175
Didier Busso, Matthieu Stierlé, Jean-Claude Thierry,
and Dino Moras

11 From No Expression to High-Level Soluble Expression in

Escherichia coli by Screening a Library of the Target Proteins
with Randomized N-Termini ............................................................ 187
Kyoung Hoon Kim, Jin Kuk Yang, Geoffrey S. Waldo,
Thomas C. Terwilliger, and Se Won Suh

12 Application of High-Throughput Methodologies

to the Expression of Recombinant Proteins in E. coli...................... 197
Yoav Peleg and Tamar Unger

13 A High Throughput Platform for Eukaryotic Genes ........................ 209

Yunjia Chen, Shihong Qiu, Chi-Hao Luan, and Ming Luo

14 High Throughput Production of Recombinant

Human Proteins for Crystallography................................................. 221
Opher Gileadi, Nicola A. Burgess-Brown, Steve M. Colebrook,
Georgina Berridge, Pavel Savitsky, Carol E. A. Smee,
Peter Loppnau, Catrine Johansson, Eidarus Salah,
and Nadia H. Pantic

15 Assembly of Protein Complexes by Coexpression

in Prokaryotic and Eukaryotic Hosts: an Overview ......................... 247
Anastassis Perrakis and Christophe Romier

16 Cell-Free Protein Synthesis for Analysis

by NMR Spectroscopy....................................................................... 257
Margit A. Apponyi, Kiyoshi Ozawa, Nicholas E. Dixon,
and Gottfried Otting

17 A Medium or High Throughput Protein Refolding Assay ............... 269

Nathan P. Cowieson, Beth Wensley, Gautier Robin,
Gregor Guncar, Jade Forwood, David A. Hume,
Bostjan Kobe, and Jennifer L. Martin
 Contents ix

18 Structural Proteomics of Membrane Proteins:

a Survey of Published Techniques and Design
of a Rational High Throughput Strategy .......................................... 277
Melissa Swope Willis and Christopher M. Koth

Section III. Biophysical and Functional Characterization of Proteins

19 Methods for Protein Characterization by Mass Spectrometry,

Thermal Shift (ThermoFluor) Assay, and Multiangle
or Static Light Scattering .................................................................. 299
Joanne E. Nettleship, James Brown, Matthew R. Groves,
and Arie Geerlof

20 High Throughput Methods for Analyzing Transition Metals

in Proteins on a Microgram Scale .................................................... 319
Anelia Atanassova, Martin Högbom, and Deborah B. Zamble

21 High Throughput Screening of Purified Proteins

for Enzymatic Activity...................................................................... 331
Michael Proudfoot, Ekaterina Kuznetsova, Stephen A. Sanders,
Claudio F. Gonzalez, Greg Brown, Aled M. Edwards,
Cheryl H. Arrowsmith, and Alexander F. Yakunin

Section IV. Structural Characterization of Proteins

22 Strategies for Improving Crystallization Success Rates ................... 345

Rebecca Page

23 Protein Crystallization in Restricted Geometry:

Advancing Old Ideas for Modern Times
in Structural Proteomics ................................................................... 363
Joseph D. Ng, Raymond C. Stevens, and Peter Kuhn

24 Fluorescence Approaches to Growing Macromolecule Crystals ..... 377

Marc Pusey, Elizabeth Forsythe, and Aniruddha Achari

25 Efficient Macromolecular Crystallization Using

Microfluidics and Randomized Design of Screening Reagents ....... 387
Andrew P. May and Brent W. Segelke

26 Increasing Protein Crystallization Screening Success

with Heterogeneous Nucleating Agents ........................................... 403
Anil S. Thakur, Janet Newman, Jennifer L. Martin,
and Bostjan Kobe

27 High Throughput pH Optimization of Protein Crystallization......... 411

Ran Meged, Orly Dym, and Joel L. Sussman
x Contents

28 Automated Structure Solution with the PHENIX Suite ................... 419

Peter H. Zwart, Pavel V. Afonine, Ralf W. Grosse-Kunstleve,
Li-Wei Hung, Thomas R. Ioerger, Airlie J. McCoy, Erik McKee,
Nigel W. Moriarty, Randy J. Read, James C. Sacchettini,
Nicholas K. Sauter, Laurent C. Storoni, Thomas C. Terwilliger,
and Paul D. Adams

29 NMR Screening for Rapid Protein Characterization

in Structural Proteomics ................................................................... 437
Justine M. Hill

30 Microcoil NMR Spectroscopy: a Novel Tool for

Biological High Throughput NMR Spectroscopy ............................ 447
Russell E. Hopson and Wolfgang Peti

31 Protein Structure Determination Using a Combination

of Cross-Linking, Mass Spectrometry, and Molecular Modeling .... 459
Dmitri Mouradov, Gordon King, Ian L. Ross, Jade K. Forwood,
David A. Hume, Andrea Sinz, Jennifer L. Martin, Bostjan Kobe,
and Thomas Huber

Section V. Structural Proteomics Initiatives Overviews

32 Structural Genomics of Minimal Organisms: Pipeline

and Results ........................................................................................ 477
Sung-Hou Kim, Dong-Hae Shin, Rosalind Kim, Paul Adams,
and John-Marc Chandonia

33 Structural Genomics of Pathogenic Protozoa: an Overview ............ 497

Erkang Fan, David Baker, Stanley Fields, Michael H. Gelb,
Frederick S. Buckner, Wesley C. Van Voorhis, Eric Phizicky,
Mark Dumont, Christopher Mehlin, Elizabeth Grayhack,
Mark Sullivan, Christophe Verlinde, George DeTitta,
Deirdre R. Meldrum, Ethan A. Merritt, Thomas Earnest,
Michael Soltis, Frank Zucker, Peter J. Myler, Lori Schoenfeld,
David Kim, Liz Worthey, Doug LaCount, Marissa Vignali,
Jizhen Li, Somnath Mondal, Archna Massey, Brian Carroll,
Stacey Gulde, Joseph Luft, Larry DeSoto, Mark Holl,
Jonathan Caruthers, Jürgen Bosch, Mark Robien, Tracy Arakaki,
Margaret Holmes, Isolde Le Trong, and Wim G. J. Hol

34 High Throughput Crystallography at SGC Toronto:

an Overview ...................................................................................... 515
Alexey Bochkarev and Wolfram Tempel

35 The Structural Biology and Genomics Platform

in Strasbourg: an Overview .............................................................. 523
Didier Busso, Jean-Claude Thierry, and Dino Moras
 Contents xi

36 Bacterial Structural Genomics Initiative: Overview of Methods

and Technologies Applied to the Process of
Structure Determination.................................................................... 537
Miroslaw Cygler, Ming-ni Hung, John Wagner, and Allan Matte

37 High Throughput Protein Production

and Crystallization at NYSGXRC .................................................... 561
Michael J. Sauder, Marc E. Rutter, Kevin Bain, Isabelle Rooney,
Tarun Gheyi, Shane Atwell, Devon A. Thompson, Spencer Emtage,
and Stephen K. Burley

38 Overview of the Pipeline for Structural and Functional

Characterization of Macrophage Proteins
at the University of Queensland ....................................................... 577
Weining Meng, Jade K. Forwood, Gregor Guncar, Gautier Robin,
Nathan P. Cowieson, Pawel Listwan, Dmitri Mouradov,
Gordon King, Ian L. Ross, Jodie Robinson, Munish Puri,
Justine M. Hill, Stuart Kellie, Thomas Huber, David A. Hume,
Jennifer L. Martin, and Bostjan Kobe

39 Structural Genomics of the Bacterial Mobile Metagenome:

an Overview ...................................................................................... 589
Andrew Robinson, Amy P. Guilfoyle, Visaahini Sureshan,
Michael Howell, Stephen J. Harrop, Yan Boucher, Hatch W. Stokes,
Paul M. G. Curmi, and Bridget C. Mabbutt

Index .......................................................................................................... 597

 Contributors

Aniruddha Achari
Raytheon IS, Huntsville, Alabama
Paul D. Adams
Berkeley Structural Genomics Center, Lawrence Berkeley National
Laboratory, and Department of Chemistry, University of California,
Berkeley, California
Pavel V. Afonine
Lawrence Berkeley National Laboratory, Berkeley, California
Margit A. Apponyi
Research School of Chemistry, Australian National University, Canberra,
Australia
Tracy Arakaki
Department of Biochemistry, University of Washington, Seattle, Washington
Cheryl H. Arrowsmith
Banting and Best Department of Medical Research, University of Toronto,
Toronto, Ontario, Canada
Anelia Atanassova
Department of Pharmacy, University of Toronto, Toronto, Ontario, Canada
Shane Atwell
SGX Pharmaceuticals, Inc., San Diego, California
Kevin Bain
SGX Pharmaceuticals, Inc., San Diego, California
David Baker
Department of Biochemistry, University of Washington, Seattle, Washington
Helen M. Berman
Research Collaboratory for Structural Bioinformatics Protein Data Bank
(RCSB PDB), Department of Chemistry and Chemical Biology,
Rutgers, The State University of New Jersey, Piscataway,
New Jersey

xiii
xiv Contributors

Georgina Berridge
The Structural Genomics Consortium, Botnar Research Centre,
University of Oxford, Oxford, United Kingdom
Alexey Bochkarev
Structural Genomics Consortium, University of Toronto,
Toronto, Ontario, Canada
Jürgen Bosch
Department of Biochemistry, University of Washington, Seattle,
Washington
Yan Boucher
Department of Chemistry and Biomolecular Sciences,
Macquarie University, Australia
Greg Brown
Banting and Best Department of Medical Research,
University of Toronto, Toronto, Ontario, Canada
James Brown
Division of Structural Biology, University of Oxford, United Kingdom
Fred Buckner
Department of Medicine, University of Washington, Seattle, Washington
Nicola A. Burgess-Brown
The Structural Genomics Consortium, Botnar Research Centre,
University of Oxford, Oxford, United Kingdom
Kyle Burkhardt
Research Collaboratory for Structural Bioinformatics Protein Data Bank
(RCSB PDB), Department of Chemistry and Chemical Biology, Rutgers,
The State University of New Jersey, Piscataway, New Jersey
Stephen K. Burley
SGX Pharmaceuticals, Inc., San Diego, California
Didier Busso
Structural Biology and Genomics Platform, IGBMC,
CNRS/INSERM/Université Louis Pasteur, Illkirch, France
Konrad Büssow
Max Planck Institute for Molecular Genetics, Department of Vertebrate
Genomics, Protein Structure Factory, Berlin, Germany
Brian Carroll
Department of Biochemistry, University of Washington, Seattle, Washington
Jonathan Caruthers
Department of Biochemistry, University of Washington, Seattle, Washington
John-Marc Chandonia
Berkeley Structural Genomics Center, Lawrence Berkeley National
Laboratory, and Department of Chemistry, University of California,
Berkeley, California
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