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Emerging Topics in
Physical
Vir logy
This page intentionally left blank
 Emerging Topics in
Physical
Vir logy

Editors

Peter G Stockley
University of Leeds, UK

Reidun Twarock
University of York, UK

Imperial College Press

ICP
Published by
Imperial College Press
57 Shelton Street
Covent Garden
London WC2H 9HE

Distributed by
World Scientific Publishing Co. Pte. Ltd.
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USA office 27 Warren Street, Suite 401-402, Hackensack, NJ 07601
UK office 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

EMERGING TOPICS IN PHYSICAL VIROLOGY

Copyright © 2010 by Imperial College Press
All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means,
electronic or mechanical, including photocopying, recording or any information storage and retrieval
system now known or to be invented, without written permission from the Publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright
Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to
photocopy is not required from the publisher.

ISBN-13 978-1-84816-464-2
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Contributors

Peter G. Stockley
Asbury Centre for Structural Molecular Biology,
University of Leeds, Leeds, LS2 9JT, UK
p.g.stockley@leeds.ac.uk
Reidun Twarock
Departments of Mathematics and Biology,
York Centre for Complex Systems Analysis,
University of York,
York, YO10 5DD, UK
rt507@york.ac.uk
Nicola G. A. Abrescia
Structural Biology Unit, CICbioGUNE,
Bizkaia Technology Park,
48160 Derio, Spain
nabrescia@cicbiogune.es
Javier Arsuaga
Department of Mathematics,
San Francisco State University,
San Francisco, CA 94132, USA
jarsuaga@sfsu.edu
Dennis H. Bamford
Institute of Biotechnology and Department of
Biological and Environmental Sciences,
Viikki Biocenter, University of Helsinki,
00014 University of Helsinki, Finland
dennis.bamford@helsinki.fi

v
vi Contributors

Robijn F. Bruinsma
Department of Physics and Astronomy,
University of California, Los Angeles,
Los Angeles, CA 90024, USA
bruinsma@physics.ucla.edu
Martin Castelnovo
Laboratoire de Physique,
Ecole Normale Superieure de Lyon,
69364 Lyon Cedex 07, France
martin.castelnovo@ens-lyon.fr
Alex Evilevitch
Department of Physics,
Carnegie Mellon University,
Pittsburgh, PA 15213, USA
alex.evilevitch@biochemistry.lu.se
Elizabeth E. Fry
Division of Structural Biology
and the Oxford Protein Production Facility,
The Wellcome Trust Centre for Human Genetics,
University of Oxford,
Headington, Oxford, OX3, 7BN, UK
Liz@strubi.ox.ac.uk
William M. Gelbart
Department of Chemistry and Biochemistry,
University of California, Los Angeles,
Los Angeles, CA 90095-1569, USA
gelbart@chem.ucla.edu
Jonathan M. Grimes
Division of Structural Biology
and the Oxford Protein Production Facility,
The Wellcome Trust Centre for Human Genetics,
University of Oxford,
Headington, Oxford, OX3 7BN, UK
jonathan@strubi.ox.ac.uk
 Contributors vii

Thomas Keef
Department of Mathematics,
University of York, York, YO10 5DD, UK
tk506@york.ac.uk
William S. Klug
Department of Mechanical and Aerospace Engineering,
University of California, Los Angeles,
Los Angeles, CA 90095, USA
klug@seas.ucla.edu
Charles M. Knobler
Department of Chemistry and Biochemistry,
University of California, Los Angeles,
Los Angeles, CA 90095-1569, USA
knobler@chem.ucla.edu
Kristopher J. Koudelka
Department of Cell Biology,
The Scripps Research Institute,
La Jolla, CA 92037, USA
koudelka@scripps.edu
Marianne Manchester
Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of California San Diego,
9500 Gilman Drive, MC 0749,
La Jolla, CA 92093, USA
mmanchester@ucsd.edu
Eric B. Monroe
Department of Microbiology,
University of Alabama at Birmingham,
Birmingham, AL 35294-2170, USA
ebmonroe@uab.edu
Alexander Yu. Morozov
Department of Physics and Astronomy,
University of California, Los Angeles,
Los Angeles, CA 90024, USA
morozov@physics.ucla.edu
viii Contributors

J. Zachary Porterfield
University of Oklahoma Health Sciences Center,
Dept of Biochemistry, Oklahoma City,
OK 73104, USA
zach-porterfield@ouhsc.edu
Peter E. Prevelige
Department of Microbiology,
University of Alabama at Birmingham,
Birmingham, AL 35294-2170, USA
prevelige@uab.edu
Neil A. Ranson
Asbury Centre for Structural Molecular Biology,
University of Leeds, Leeds, LS2 9JT, UK
n.a.ranson@leeds.ac.uk
Janne J. Ravantti
Institute of Biotechnology and Department
of Biological and Environmental Sciences,
Viikki Biocenter, University of Helsinki,
00014 University of Helsinki, Finland
janne.ravantti@helsinki.fi
Joaquim Roca
Instituto de Biologia Molecular de Barcelona,
CSIC, Barcelona, Spain
joaquim.roca@ibmb.csic.es
Wouter H. Roos
Fysica van complexe systemen,
Natuur- en Sterrenkunde, Vrije Universiteit,
1081 HV Amsterdam, The Netherlands
wroos@few.vu.nl
Joseph Rudnick
Department of Physics and Astronomy,
University of California, Los Angeles,
Los Angeles, CA 90024, USA
jrudnick@physics.ucla.edu
 Contributors ix

David I. Stuart
Division of Structural Biology
and the Oxford Protein Production Facility,
The Wellcome Trust Centre for Human Genetics,
University of Oxford,
Headington, Oxford, OX3 7BN, UK
dave@strubi.ox.ac.uk
De Witt Sumners
Department of Mathematics,
Florida State University,
Tallahassee, FL 32306-4510, USA
sumners@math.fsu.edu
Gijs J. L. Wuite
Fysica van complexe systemen,
Natuur- en Sterrenkunde, Vrije Universiteit,
1081 HV Amsterdam, The Netherlands
gwuite@nat.vu.nl
Adam Zlotnick
Department of Biology,
Indiana University,
Bloomington, IN 47405, USA
azlotnic@indiana.edu
This page intentionally left blank
Preface

Emerging Topics in Physical Virology is a state-of-the-art account of recent

developments in the analysis and modelling of virus structure, function and
dynamics. It pays tribute to the importance of interdisciplinary research by
integrating an exposition of experimental techniques such as cryo-electron
microscopy, atomic force microscopy and mass spectrometry with mathe-
matical and biophysical modelling techniques. The number of chapters co-
authored by experimentalists and theoreticians is testimony to the impor-
tance of interdisciplinarity in tackling some of the most challenging and
exciting research problems in this area.
The aim of this book is to introduce the reader to recent develop-
ments in the field, and to provide a comprehensive review of the results
that prompted them. It is therefore not only a primer for researchers work-
ing in the analysis and modelling of viruses, but also serves as an intro-
duction for non-experts into this tantalising field of research. The book
starts with a description of cryo-electron microscopy by Neil Ranson and
Peter Stockley and demonstrates its power in determining the structure
and dynamics of viruses. Structural insights gained from X-ray crystallog-
raphy have revealed an intriguing phenomenon: there is a striking conser-
vation in the topologies of the capsid proteins that form the containers
encapsulating viral genomes. This has prompted Dennis Bamford, David
Stuart and collaborators to classify viral families into lineages based on
the concept of the viral ‘self’. An important feature of this conservation
of capsid protein folds is that it appears to be non-sequence-specific, i.e.
the chemical structures of proteins with homologous folds can often be
very different. This suggests that there must be a guiding principle for the
formation of the capsid proteins that is independent of their sequences.
Thomas Keef and Reidun Twarock suggest that the icosahedral symmetry
of many viruses may provide such a guiding principle, and they introduce
novel group theoretical techniques to model this effect. Their approach
implies that a wide spectrum of viral features can be predicted based on
symmetry, and that perhaps the limited number of structural folds is a

xi
xii Preface

consequence of the fact that only a limited number of layouts are possible
for viruses with such symmetry.
Atomic force microscopy provides important insights into the
mechanical properties of viruses as detailed in the chapter by Wouter Roos
and Gijs Wuite. The authors discuss capsid shell structure, presence of
encapsidated material, capsid failure, maturation and capsid protein muta-
tions in relation to viral material properties and highlight similarities and
differences for different types of viruses. Another important experimen-
tal technique in the study of viruses is mass spectrometry. Eric Mon-
roe and Peter Prevelige show how this technique can be used to gain
invaluable information on viral proteins. Mass spectrometry can also play
a crucial role in the study of virus assembly, i.e. the process of forma-
tion of viral particles from their protein building blocks and genomic
material. An overview of capsid assembly kinetics is provided by one of
the pioneers in this area, Adam Zlotnick, and his collaborator Zachary
Porterfield. Their chapter covers both modelling and experimental tech-
niques and provides a comprehensive overview of viral capsid kinetics. An
important factor in virus assembly and disassembly is the mechanical stress
on different components of the viral capsid. A beautiful account of how
stress distributions impact on the assembly and disassembly of viral capsids
formed from pentamers and hexamers is provided by Robijn Bruinsma and
collaborators.
Viruses may package genomic material in the form of DNA or RNA.
An important question concerning the formation of RNA viruses is ‘what
determines the size of an RNA virus?’ It is addressed by Chuck Knobler
and Bill Gelbart in their discussion of the correlation between capsid and
genome sizes. The impact of genome length versus capsid size on the
physics of viral infectivity is also discussed with respect to double-stranded
DNA (dsDNA) phages by Alex Evilevitch and Martin Castelnovo.
Another intriguing feature of packaged DNA genomic material is its
topology. Together with their experimental collaborator Joaquim Roca,
Jarvier Arsuaga and De Witt Sumners provide a comprehensive account
of the mathematical and experimental analysis of the topology of
viral DNA.
A volume on emerging topics in physical virology would not be com-
plete without a discussion of the fascinating applications of viruses and
virus-like particles in biomedical nanotechnology that are opened up by
 Preface xiii

these recent theoretical and experimental approaches. We therefore con-

clude with a chapter by Kristopher Koudelka and Marianne Manchester
that discusses the state of the art in this area.
We would like to express our special thanks to the authors of these fas-
cinating chapters for making this volume possible by sharing their exciting
research with us.

Peter Stockley and Reidun Twarock

March 2009
This page intentionally left blank
Contents

Contributors v

Preface xi

Chapter 1: Cryo-Electron Microscopy of Viruses 1

Neil A. Ranson and Peter G. Stockley
Chapter 2: What Does it Take to Make a Virus:
The Concept of the Viral ‘Self’ 35
Nicola G. A. Abrescia, Jonathan M. Grimes,
Elizabeth E. Fry, Janne J. Ravantti,
Dennis H. Bamford and David. I. Stuart
Chapter 3: Beyond Quasi-Equivalence: New Insights
Into Viral Architecture via Affine Extended
Symmetry Groups 59
Thomas Keef and Reidun Twarock
Chapter 4: Mechanical Properties of Viruses 85
Wouter H. Roos and Gijs J. L. Wuite
Chapter 5: Investigating Viral Structure, Function
and Dynamics with Mass Spectrometry 103
Eric B. Monroe and Peter E. Prevelige
Chapter 6: An Overview of Capsid Assembly Kinetics 131
J. Zachary Porterfield and Adam Zlotnick
Chapter 7: Assembly and Disassembly of Deltahedral
Viral Shells 159
Alexander Yu. Morozov, Joseph Rudnick,
Robijn F. Bruinsma and William S. Klug
Chapter 8: What Determines the Size of an RNA Virus? 185
Charles M. Knobler and William M. Gelbart

xv
xvi Contents

Chapter 9: Physics of Viral Infectivity: Matching Genome

Length with Capsid Size 217
Alex Evilevitch and Martin Castelnovo
Chapter 10: Topology of Viral DNA 255
Javier Arsuaga, Joaquim Roca
and De Witt Sumners
Chapter 11: The Use of Viruses in Biomedical Nanotechnology 289
Kristopher J. Koudelka
and Marianne Manchester
Index 313
Chapter 1

Cryo-Electron Microscopy of Viruses

Neil A. Ranson∗,† and Peter G. Stockley∗,‡

Cryo-electron microscopy (cryo-EM) is a structural technique that images

biological macromolecules in native-like conditions, and has been widely
applied to the study of viruses. Virus structures have been determined by
cryo-EM at resolutions ranging from molecular (∼30–50 Å) to near-atomic
(∼4 Å). Here we introduce cryo-EM of virus particles for the non-expert
reader and review how some of the key cryo-EM studies have advanced our
understanding of virus biology. We also describe the latest advances in cryo-
EM. These advances are on the one hand driving cryo-EM studies of sym-
metric viruses towards atomic resolution. On the other, they are developing
structural methods that allow the study of individual, pleiomorphic virus
particles and the interactions they make with cellular machinery.

1. Introduction
The molecular details of how viruses infect and hijack the cellular processes
of their host cells are of critical importance in biology and medicine. It
is only through a precise understanding of such events that new anti-viral
therapies will be developed. One of the key elements of this growing under-
standing of the viral life cycle has been an increased understanding of the
structure of viruses, and in particular their dynamic properties. Together
with X-ray crystallography, cryo-electron microscopy (cryo-EM) tech-
niques have played a central role in studying virus structure. At the same
time, viruses have played an equally important role in the development
of cryo-EM and single-particle image processing as techniques in modern
structural biology. In this chapter we will illustrate these developments for
non-expert readers with selected examples.

∗ Asbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2

9JT, UK. E-mails: † n.a.ranson@leeds.ac.uk; ‡ p.g.stockley@leeds.ac.uk

1
2 N. A. Ranson and P. G. Stockley

Fig. 1. Electron micrograph of Turnip Yellow Mosaic Virus (TYMV) negatively

stained with 1% phosphotungstic acid. From Brenner & Horne (1959). Biochem. Bio-
phys. Acta. 34, p. 103.

Electron microscopy has been used to image viruses for more than
50 years. Initial studies involved the use of negative staining, in which the
virus is placed on a continuous carbon film, coated in a heavy metal salt
such as uranyl acetate, ammonium molybdate or phosphotungstic acid,
and then dried before imaging (Fig. 1). Such treatment embeds the virus
in a cast of the heavy metal salt and it is this cast, rather than the biolog-
ical macromolecule itself, that gives rise to contrast in the images; hence
‘negative’ staining. Staining methods are rapid, generate high image con-
trast, and allow relatively large electron doses to be used, producing readily
interpretable relatively low-resolution images of virus particles. However,
staining also has major disadvantages. Firstly, penetration of stain into the
interior of macromolecules is limited by both the structure of those macro-
molecules and by the size of the stain crystals. In practice this means that
negative staining typically reveals only the external envelope of a macro-
molecule rather than any internal features. Secondly, the drying process
can significantly distort the structure being imaged, as specimens are typi-
cally flattened onto the carbon support film as they dehydrate (see Fig. 2a).
Such structural deformations are exacerbated by the pH and ionic strength
of the stain solution, which places the biological macromolecule in a pro-
foundly non-native environment. In summary, negatively stained images of
viruses therefore are excellent at identifying the presence of viral particles
in both purified and cellular samples, and this remains a major tool in the
search for the presence of novel viruses in tissues.
 Cryo-Electron Microscopy of Viruses 3

Fig. 2. A schematic representation of specimens for EM studies. (a) A negatively-

stained virus (blue hexagon) adsorbed to a carbon support film (grey), and embedded
in deep stain (orange). Note the flattening of the hexagonal virus onto the support
film. (b) A viruss particle partially embedded in shallow stain. The portion of the virus
structure not embedded in stain will not contribute to images of the specimen. (c) A
cryo-EM specimen. Virus particles are unstained and trapped in a layer of vitreous ice.
The ice layer is formed in a hole in the carbon support film.

A further complication of electron microscopy in general is caused

by the nature of image formation in the microscope. EM has a depth
of field that is significantly larger than most biological specimens. The
consequence of this is that the images formed are projections of the three-
dimensional (3-D) object onto the plane of the two-dimensional (2-D)
recorded image, which greatly complicates interpretation. Various tricks
have been developed to reveal information about the third dimension.
One technique often used is metal shadowing at defined angles, which
produces a pattern of electron dense material around particles of interest.
The lengths of shadowed areas are proportional to the height of the object.
More sophisticated image processing techniques have also been developed
to reconstruct 3-D objects from a series of 2-D projection images, tech-
niques that are very similar to those used in computed tomography (CT)
scanning in medicine. Such methods work particularly well for viruses
that have highly symmetric capsids. A major advance has been the use of
cryogenic freezing of unstained samples (cryo-EM) and techniques for
data collection that allow 3-D reconstructions from samples undamaged
by excessive exposure to the electron beam (see below). Such techniques
yield structures for viruses at resolutions that can rival those of X-ray crys-
tallography, and of course do not require crystallisation of the samples
before structure determination. Fortunately it turns out that in most cases
to date structures determined by X-ray and cryo-EM methods are very sim-
ilar, and X-ray structures can often be fitted into cryo-EM density. This
implies that crystal structures reflect viral structures in solution but it has
also opened up a wonderful synergy between the two techniques, as there
are many conditions in which virologists would like to examine viruses
4 N. A. Ranson and P. G. Stockley

that are unlikely ever to be accessible by single crystal diffraction studies.

Recent developments allow 3-D reconstructions to be determined for sin-
gle virus particles, further extending the power of cryo-EM to interrogate
viral life-cycles. Such techniques allow the determination of tomograms
of viruses that lack isometric structures, such as the major pathogens of
influenza, HIV and herpes. Viral asymmetry is therefore no longer a barrier
to structural studies

2. The Cryo-EM Technique

As a result of the problems described above with interpreting stained
images, structural biologists were keen to move to imaging of unstained
specimens. The development of cryo-EM built upon the long estab-
lished concept that low temperature preserves biological specimens. How-
ever at the molecular level, the formation of ice crystals during freezing
can disrupt biological structures and withdraws water molecules from their
hydration shell causing macromolecules to become partially dehydrated.
Furthermore, crystalline ice diffracts the electron beam, preventing use-
able image formation. Dubochet and colleagues at the EMBL in Heidel-
berg discovered that if a biological specimen was frozen sufficiently rapidly
the formation of ice crystals was prevented, preserving the solution con-
formation and allowing it to be imaged in a thin layer of vitreous ice. The
key to this rapid freezing was the use of liquid ethane or propane, cooled to
near liquid nitrogen temperatures, to freeze the samples. These coolants
have extremely high thermal conductivity, which allow extremely rapid
cooling rates to be achieved. Together with the low mass of an aqueous
thin film, cooling rates approaching a million degrees per second can be
achieved during vitrification. In the vitreous ice layer that results from such
rapid cooling, biological macromolecules remain hydrated, and in a struc-
tural state essentially identical to that seen in the liquid phase. Typically,
the thin film of ice was formed and frozen in a hole in a carbon support
film, ameliorating the effects of a support film that can flatten particles and
cause preferred orientations to be observed (see Fig. 2). The first practical
results of this new technique were images of viruses: unstained, frozen-
hydrated Semliki Forest Virus and bacteriophage T4 (see Fig. 3e). Even in
these first cryo-EM images, the potential of the technique for studying the
structure of viruses was immediately apparent. The images of SFV imme-
diately settled an ongoing debate about whether SFV had a T = 3 or a
 Cryo-Electron Microscopy of Viruses 5

Fig. 3. Cryo-EM sample preparation. (a) A cryo-EM freezing apparatus. An EM grid

is held in forceps above a reservoir of liquid nitrogen-cooled liquid ethane. Sample in
water of buffer is added, excess liquid is blotted away (here by pneumatically-driven
blotters) and then the grid is plunged into the liquid ethane reservoir. (b) A typical EM
grid The grids are ∼3 mm in diameter and consist of a mesh made from a variety of
metals, and in various spacings. (c) Grids covered with a support film lithographically
etched to contain a regular array of regularly sized holes are commercially available.
The support film shown is a Quantifoil R2/2 grid (Quantifoil Microtools, Gmbh), in
which the holes are 2 µm in diameter). (d) A close-up view (∼1200x) of such a support
film with a thin layer of vitreous ice in the EM is also shown. (e) The first published
cryo-EM images of a virus; Semliki Forest virus. From Adrian et al., (1984).
6 N. A. Ranson and P. G. Stockley

T = 4 capsid morphology, unambiguously revealing a T = 4 morphology

(Adrian et al., 1984).

3. Determining the 3-D Structure of Viruses

from EM Data
Like all methods that are in principle suitable for determining the high-
resolution structure of biological macromolecules, electron microscopy
has both advantages and disadvantages. Perhaps the most notable advan-
tage of an electron microscope might sound trivial but is not; an electron
microscope records an image. The process of image formation means that
both amplitude and phase information are recorded simultaneously, in
contrast to diffraction-based methods where only amplitudes are captured,
and phase information has to be derived. However, aberrations in the mag-
netic lenses used to focus electrons (which allows formation of the image)
mean that very small apertures are required in electron microscopes. Small
apertures contribute to the large depth of field in EM. Together with the
penetrating nature of electrons, this results in the 3-D electron density
of the object being projected onto the 2-D plane of the image (c.f. a
medical X-ray). The fact that EM generates projection images of biolog-
ical structures means that it is extremely difficult to interpret individual
EM images. However, it also means that true 3-D structural information
is accessible. 3-D reconstruction of an object from projections is possi-
ble owing to the fact that the Fourier transform of a projection image
is a central section of the 3-D Fourier transform of the original object
(Fig. 4).
Thus, if all possible views of the object are available, then a full 3-D
reconstruction of the object is possible, in principle to the resolution limit
of the instrument. For the case of electrons in the 2–300 keV range as
used in modern microscopes, this is better than 1 Å resolution. The key
problem faced in calculating such a 3-D structure is determining in which
orientation the individual projections of the original object were imaged.
The orientation of the projected structure defines which central section
through the 3-D transform of the object the projection represents. From
this it can readily be seen that a full range of all possible orientations leads
to the most completely sampled 3-D transform, and hence contributes
towards higher resolution. The essence of determining a virus structure
 Cryo-Electron Microscopy of Viruses 7

Fig. 4. The principle of how to calculate a 3-D structure from 2-D projections. The
virus is randomly oriented in vitreous ice layer of a cryo-EM grid. The recorded images,
containing noise and distorted by the effects of the microscope CTF are projections
of different views of the virus onto the plane in which the image is recorded. The
2-D Fourier transform of those images are central sections through the 3D Fourier
transform of the virus. Knowing the orientation of each particle tells you which central
section of the 3-D transform that virus particle represents — i.e. how the slices fit
together. The degree to which the 3-D transform is populated is one of the limits on
resolution in cryo-EM studies; hence the need for randomly distributed orientations
and the advantage of high symmetry. The 3-D object is reconstructed by reverse Fourier
transformation to give a 3-D electron density map.
8 N. A. Ranson and P. G. Stockley

by cryo-EM is therefore simply to determine the orientation of each pic-

ture of the virus with as high a degree of accuracy as possible. Details
of exactly how the orientation of each imaged virus particle is found are
beyond the scope of this article. However, in essence a number of different
approaches are possible which share the same basic idea that experimen-
tal images of the virus are compared to reprojections of a model struc-
ture for the virus. For review, see (Baker et al., 1999; van Heel et al.,
2000).
Implicit in the idea that all possible views are required is the fact that
structures of highly symmetric particles are more readily calculated, as the
symmetry of the particle helps to ensure that all possible views are sampled.
Averaging of symmetry-related views also increases the effective size of
datasets, meaning that fewer raw images are required for a given resolution
than for asymmetric particles. Symmetry averaging does however ‘average-
away’ any asymmetric features of a virus, such as single-copy infectivity or
maturation proteins which are commonly essential for viral lifecycles.
The negative-staining method that dominated EM imaging of viruses
until the mid-1980s presented major challenges to the application of this
approach to 3-D structure determination. Very often biological particles
adhere to the carbon support film on the EM grid, deforming the native
structure and resulting in preferred particle orientations that make 3-D
structure determination difficult because these do not contain enough
information. For highly symmetric particles such as isometric viruses, this
is not an insurmountable problem, as symmetry ensures an even cover-
age/sampling of the 3-D transform of the reconstructed density. How-
ever, the size of such particles means that obtaining images of viruses fully
embedded in stain is extremely difficult, and for larger viruses essentially
impossible. Since it is the interaction of electrons with the stain layer that
generates contrast, this results in the information from un-embedded por-
tions of the virus not being recorded in the image, i.e. the images represent
projections of only part of the structure (see Fig. 2b). Furthermore, owing
to the problems associated with staining, only information on the surface
envelope of the virus is obtained.
Preservation and imaging of the entire native-like structure is the clear
advantage of cryo-EM over staining methods. However, the fact that the
specimen is unstained results in a different set of problems for structure
determination. Firstly, amplitude contrast in cryo-EM images arises from
the scattering power of the atoms found in the specimen, which is related
 Cryo-Electron Microscopy of Viruses 9

to their mass. Hence the use of heavy metal salts in negative staining. The
masses of atoms typically found in proteins and/or nucleic acids are very
similar to those found in the surrounding water and buffer molecules of
the vitreous ice layer. This means that although cryo-EM images contain
information from the entire macromolecular structure, they typically have
extremely low contrast. This problem is made markedly worse by the radi-
ation sensitivity of unstained biological macromolecules, requiring that
the electron dose delivered to the specimen be kept low to minimise radi-
ation damage. Together these factors result in the characteristically poor
signal to noise ratios (SNR) of cryo-EM images, and necessitate compu-
tational averaging of data to improve the SNR to a point where structure
determination is possible. In essence, in cryo-EM noisy images of indi-
vidual molecules have to be explicitly averaged after the data is recorded,
whilst in X-ray diffraction averaging is an intrinsic property of the crystal.
This averaging creates significant computational challenges that need to
be overcome in order to determine cryo-EM structures, especially to high
resolution.
To a limited extent, the problem of low amplitude contrast in cryo-EM
images can be overcome by tuning the optical properties of the micro-
scope to introduce a second type of contrast into the recorded images:
phase contrast. In practice, this is routinely achieved by defocusing the
microscope, which introduces low-resolution phase contrast that is often
essential to allow relatively small objects, such as virus capsids, to be found
in noisy micrographs. It also means that distortion of the observed image
by the microscope’s contrast transfer function (CTF) becomes significant
and problematic (Fig. 5). A CTF is a phenomenon, common to all opti-
cal systems, that defines how information is transferred as a function of
spatial frequency (i.e. resolution) in Fourier space. The form of this func-
tion is of an oscillating sinusoidal variation in information transfer with
increasing frequency and decreasing amplitude (Fig. 5f). In real space
(i.e. in the observed image) the effects of a CTF are to convolute the
image of the object being studied with a point spread function (PSF). A
PSF spreads information from each area of the image into surrounding
areas and attenuates information at high resolution. The practical con-
sequences of using highly defocused images are therefore that although
they have more low-resolution contrast, allowing the particles to be found,
the particles themselves have less high resolution information and are
profoundly distorted, necessitating computational CTF correction during
10 N. A. Ranson and P. G. Stockley

Fig. 5. Contrast transfer functions in cryo-EM. (a) The effects of a CTF on the
observed image. The 3-D structure (a) is projected (in a computer) to give a 2-D
projection image with no added CTF (b). The effects of a CTF function are then
simulated with defocus of 1.0 µm (c), 2.0 µm (d), and 4.0 µm (e). The effects of the
Fourier space CTF function on the real image are of a point spread function, which
spreads information from the particle into surrounding areas. (f) Plots of typical CTF
functions applied in (c–e). A CTF has an oscillating form with increasing frequency
and decreasing amplitude — i.e. there are contrast reversals, spatial frequencies where
no information is transferred (‘zeroes’) and attenuation of high-resolution informa-
tion. Only by combining data at different degrees of defocus can full restoration of
information at all resolutions by achieved.

the structure refinement calculations. Such corrections essentially flip the

image contrast in regions where the CTF has made it negative, removing
the effects of the PSF.

4. Complementarity between Cryo-EM

versus X-ray Methods
Advances in all aspects of X-ray crystallography, especially in robotic crys-
tallization condition screening, and in the advent of synchrotron radiation
sources, have led to ever larger and more complicated macromolecular
 Cryo-Electron Microscopy of Viruses 11

complexes being solved by X-ray methods. Such advances are exemplified

by recent X-ray structures of large enveloped viruses such as PRD1
and PM2 (Cockburn et al., 2004; Abrescia et al., 2008). Despite such
remarkable successes, generally speaking the larger the virus the more
difficult it is to grow crystals that diffract to high resolution. Cryo-EM,
which, as described above, has no requirement for crystallization, is appli-
cable (and has been applied) to all classes of virus from the smallest (plant
satellites) to the largest (mimivirus). Its use has been especially power-
ful in the study of larger viruses (especially those containing lipid bilayer
envelopes), virus-receptor complexes, and transient states in virus matu-
ration pathways. Some notable studies in these areas are described in the
following sections.
A recurring theme in these studies is the complementarity between
X-ray and cryo-EM techniques. Cryo-EM maps at even quite modest
resolutions have been used to phase X-ray data. Fitting of X-ray coor-
dinates into low or intermediate resolution density dramatically enhances
the interpretable information content of the cryo-EM structure. X-ray and
cryo-EM methods have also been used extensively to map different states
in the same maturation pathway.

5. Structure of Large Enveloped Viruses

Several very large viruses have been isolated and their structures deter-
mined by cryo-EM. Chilo iridescent virus (CIV) and Paramecium bur-
saria chlorella virus type 1 (PBCV-1) infect very different hosts (insect
and unicellular green algae respectively) but have strikingly similar struc-
tural features (Yan et al., 2000). CIV, with a diameter of ∼1850 Å and
a T = 147 morphology, is marginally smaller than PBCV-1 (diameter
1900 Å, with a T = 169d morphology). An even bigger virus Phaeocystis
pouchetii virus (PpV01) has a capsid with T = 219d quasi-symmetry and
diameter of ∼2200 Å (Yan et al., 2005). However even these giant viruses
are dwarfed by another virus discovered at around this time. Mimivirus was
first isolated from amoeba in a water-cooling tank, and initially thought to
be a bacterium (it is ∼3x the size of the smallest known bacterium). The
capsid is currently thought to have a T =∼1179 morphology (Fig. 6; Xiao
et al., 2005; Xiao et al., 2009), and packages a dsDNA genome of ∼1.2Mb
encoding 911 proteins (Raoult & Forterre, 2008). The smallest known
12 N. A. Ranson and P. G. Stockley

Fig. 6. Extremes of virus size. (a) X-ray structure of Satellite Panicum Mosaic
virus (SPMV; Ban & McPherson, 1995), amongst the smallest of known virus par-
ticles. SPMV has a T = 1 morphology and a diameter of ∼160 Å. (b) The largest
known virus: Mimivirus. Mimivirus has a T ∼= 1179 morphology and a diameter of
∼5000 Å. From Xiao et al., 2005; Xiao et al., 2009. Inset in the small box is SPMV
shown at the same scale as mimivirus.

free-living organism is a mycoplasma and its genome encodes just 470 pro-
teins. Mimivirus therefore blurs the boundaries between viruses and uni-
cellular organisms. Indeed, mimivirus has since been reported to be itself
parasitised by a smaller virus, the first ‘virophage’ (La Scola et al., 2008).

6. Virus — Receptor Interactions

The interaction of a virus with its cellular receptor is of critical impor-
tance in infection, and cryo-EM has played a significant role in elucidat-
ing the molecular details of such interactions. One area in which this
has been especially apparent is in the binding of rhinovirus, a picor-
navirus, to intracellular adhesion molecule-1 (ICAM-1). These studies
helped to address a fundamental question regarding virus–receptor inter-
actions: viruses are known to mutate rapidly, helping them to evade the
host immune response, yet make specific interactions with their recep-
tors. It is, however, difficult to rationalise how such rapid mutation, and
the accompanying structural changes that it promotes, can be compatible
with the maintenance of a stable, specific interaction with a host molecule.
Kolatkar et al. (1999), showed how soluble fragments of ICAM-1 bound
 Cryo-Electron Microscopy of Viruses 13

in a canyon on the surface of the virus. This surface feature was thought
to be too small to allow antibody access, providing spatial separation of
the receptor-binding site from the immune response. Surface residues
could therefore mutate more rapidly, allowing immune evasion, whilst the
receptor-binding site was maintained in its canyon. Although, antibody
binding has now been detected to epitopes within the canyon, the basic
proposal remains a paradigm for picornavirus — receptor interactions.
Structural studies on poliovirus, another picornavirus, have visu-
alised the virus bound to its cellular receptor incorporated into lipo-
somes (Bubeck et al., 2005). Such a structure represents a virus that is
poised to deliver its genomic information across the liposome bilayer,
and provides invaluable information about the process of infection via
membrane-embedded receptors. The authors first determined the viral
site at which receptor binding occurred using a novel post-imaging fidu-
cial marker technique. Briefly they determined the orientation of each
image in their dataset, and then added a white ‘spot’ to each image
marking the attachment site. When such ‘spotted’ images were used in
an icosahedral reconstruction a clear density above each five-fold ver-
tex was observed, unambiguously showing that the virus binds its intact
membrane-embedded receptor at a five-fold vertex. In turn this knowl-
edge allowed a reconstruction of the virus-receptor-liposome complex to
be calculated using C5 (five-fold rotational averaging around the attach-
ment site) symmetry (Fig. 7). The resulting five-fold averaged structure of
the complex shows how receptor binding brings one of the twelve five-fold
vertices of the capsid into close proximity with the membrane of the lipo-
some, where the lipid bilayer is perturbed. Presumably this perturbation
is the first step in allowing the viral RNA to access the interior of the host
cell. The same authors have now applied similar methodologies to examine
the membrane-attachment complex of Semliki forest virus (SFV), a model
for enveloped virus fusion. SFV also approaches the membrane along a
five-fold axis that at least suggests this may be a generic feature of receptor
recognition and fusion events.

7. Maturation Processes
Another area of virology in which cryo-EM has made a substantial con-
tribution to our expanding knowledge is in viral maturation. Although
significant efforts have been directed at HSV-1 nucleocapsid maturation,
the most complete description of a viral maturation pathway to date is for
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