Table Of ContentProtein Crystallography
Challenges and Practical Solutions
Chemical Biology
Editor-in-chief:
Tom Brown, University of Oxford, UK
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Sabine Flitsch, University of Manchester, UK
Nick J. Westwood, University of St Andrews, UK
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1: High Throughput Screening Methods: Evolution and Refinement
2: Chemical Biology of Glycoproteins
3: Computational Tools for Chemical Biology
4: Mass Spectrometry in Chemical Biology: Evolving Applications
5: Mechanisms of Primary Energy Transduction in Biology
6: Cyclic Peptides: From Bioorganic Synthesis to Applications
7: DNA-targeting Molecules as Therapeutic Agents
8: Protein Crystallography: Challenges and Practical Solutions
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Protein Crystallography
Challenges and Practical Solutions
Edited by
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Imperial College London, UK
Email: [email protected]
and
Gwyndaf Evans
Diamond Light Source, UK
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FOREWORD
Protein Crystallography:
Faster, Smaller, Stronger
DAVID I. STUART* AND JAMES H. NAISMITH
*Email: [email protected]
The symmetry and beauty of crystals, found commonly as minerals in
rocks and of course in the production of salt, has long been a source of
fascination. The underlying determinant of crystal morphology emerged
when the work of Ernest von Laue revealed that crystals diffracted X-rays
and Lawrence Bragg working alongside his father William derived a for-
mula which explained the phenomenon. Since X-rays were being scattered
by electrons, it was realised that these diffracted beams held clues to the
underlying arrangements of the atoms and thereby the structure of the
material. Lawrence Bragg in his Nobel Lecture noted ‘‘The examination of
crystal structure, with the aid of X-rays has given us for the first time an
insight into the actual arrangement of the atoms in solid bodies.’’
Presciently, he noted ‘‘There seems to be hardly any type of matter in the
condition of a true solid which we cannot attempt to analyse by means of
X-rays.’’ In these early days, the structures ‘determined’ by crystallography
were simple salts. Since it was already known that many organic and
inorganic compounds could be crystallised, the need to develop robust
methods to recover the relative phases of the diffracted beams was plain,
if more complex structures were to be solved. Progress continued, with
notable early landmarks in 1929; Kathleen Lonsdale’s proof that benzene
rings were flat and John (JD) Bernal’s correct structure of cholesterol.
Almost simultaneously, James B. Sumner had established beyond doubt
that enzymes were a single defined chemical species that could be purified
ChemicalBiologyNo.8
ProteinCrystallography:ChallengesandPracticalSolutions
EditedbyKonstantinosBeisandGwyndafEvans
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v
vi Foreword
the same as any other chemical substance. Sumner grew crystals of urease
and concanavalin A isolated from jack bean; his work inspired others to
use crystallisation as an increasingly routine means of purifying proteins
(and establishing their purity). Significant numbers of proteins were being
crystallised by biochemists.
The advances in these fields of crystallography and protein biochemistry
continued essentially in parallel. Up to the development of direct methods
begun in 1953 by Herbert Hauptman and Jerome Karl, the solution of so-
called small molecule crystal structures involved time consuming Patterson
methods,placementofknown fragments,isomorphous series,heavyatoms
and intuition. As direct methodsexpanded in power and utility,the solving
of the structure of small molecules by crystallography became increasingly
routine. The process was dramatically accelerated by the increasing
availabilityandpowerofcomputers.Thepowerofsupercomputerswasseen
in the early 1950’s when Dorothy Crowfoot Hodgkin and Jenny Glusker,
workingonthestructureofvitaminB12,calledinKenTruebloodatUCLAto
accelerate the determination of the structure by ‘post and cable’ support
fromhislocalmachine(whichwasofcoursemillionsoftimesslowerthana
smartphone).
From its inception, protein crystallography has involved physicists,
chemists and biologists working at the interface of their disciplines.
Working with J. D. Bernal, Hodgkin in 1934 showed that protein crystals,
like those of common salt, diffracted X-rays according to Bragg’s law.
A key experimental constraint was that the protein crystals had to be kept
hydrated. Max Perutz, a PhD student encouraged by both J. D. Bernal and
Lawrence Bragg, began to study crystals of haemoglobin by X-ray dif-
fraction. Interrupted by the Second World War, Perutz resumed his work
and showed that by replacing a water molecule with a much heavier atom
such as mercury, it was possible to experimentally determine the phases
and thus the structure of haemoglobin. His colleague John Kendrew used
the same approach to determine the structure of myoglobin. In 1959, these
structures were available around 5.5 and 2 Å respectively and provided
unprecedented insights into the molecular mechanism of oxygen binding
and transport.
Others followed suit, Hodgkin, already a Nobel Prize winner for her
work on penicillin and vitamin B12, determined the structure of insulin in
1969,DavidPhilipslysozymein1965andDavidBlowchymotrypsinin1967.
In each case, these structures revolutionised our understanding of the
molecular basis of biology. As Perutz said, ‘‘I think we pushed knowledge
of living matter down to the atomic level and this enormously increased
our depths of understanding of the nature of life.’’ This power was widely
accepted by 1970. A method for structure solution using related structures
emerged in the early 1970’s. This, the molecular replacement method, is
now used for most structural analyses of new protein structures and
was developed following first suggestions by Walter Hoppe, and then
Michael Rossmann and David Blow. Viruses, DNA and RNA oligomers, and
ProteinCrystallography:Faster,Smaller,Stronger vii
membrane proteins all were shown to be crystallisable and thus be
amenable to study. The simple act of crystallising viruses was a major
landmark in virology. In 1935 Wendell Stanley crystallized tobacco mosaic
virus, which led to a share of a Nobel prize, the first awarded to virology.
This of course was many years before structure determination was feasible,
but it was clear that Bragg’s prediction of the ubiquity of crystal structure
analysis was true. During the 1970’s the technique became widely
established, although new structures came slowly.
The utility of protein crystallography in the 1970’s and early 1980’s was
limited by significant technical challenges. Structure solutions were hard
won following years of trial and error experiments; data collection moved
to photographic film, which was time consuming; X-ray sources were weak
(access to powerful synchrotrons was not yet routine); only the most
abundant proteins were plausible candidates for isolation; many proteins
only gave small crystals and when these crystals could be examined at
synchrotrons they often died during data collection. In a period of about
five years from the mid 1980’s each of these problems was overcome by
what individually could be characterised as incremental advances but
when combined were transformative. Perhaps the greatest single impact
was the application of recombinant DNA technology. The principles were
discovered in the 1970’s and the development of methods over subsequent
years eventually opened up even the most recalcitrant human proteins to
crystallographic analysis. This allowed structural biologists to focus on
the biological significance of a target, rather than its natural abundance.
Steady advances in molecular biology now make overexpression and
simple purification routine, with the structural genomics/proteomics
post-millennium efforts putting structural biologists centre stage in the
development of protein production methods. Freezing of crystals before
data collection greatly reduced their decay during exposure to X-rays
improving data quality, new beamlines at synchrotrons came on stream
giving much more access, machine readable image plates replaced film
greatly speeding up and improving data collection and the power and
availability of small computers meant that even small groups could solve
structures quickly in their own laboratory. Increased intensity and better
focus of synchrotron beams made it possible to study much smaller crys-
tals. The replacement of methionine with selenomethionine, pioneered
by Wayne Hendrickson, became a generic vehicle, making phase de-
terminationusing anomalousscatteringsignalsalmostroutine,accelerated
by access to tuneable high energy synchrotron beams (in the UK this was
helped by the MRC’s purchase, with EMBL, of BM14 at the ESRF). In the
1990’s the number of structures being determined exploded. By this point
all major pharmaceutical companies were using the technique in drug
discovery and most biological problems were being advanced by structural
study at some level. In the years following 2000, industry and structural
genomics/proteomics consortia drove a further round of advances, notably
standardisation which facilitated automation, remote access and near real
viii Foreword
time data analysis. Then in 2011 a new chapter opened with the first
publications of results from a hard X-ray free electron laser (XFEL), the
ultimate impact of XFEL on protein crystallography is still an open
question, and is discussed in the current volume.
The power and scope of the technique continues to grow and although
cryo electron microscopy has dethroned crystallography for larger as-
semblies, it remains crucial for many different approaches. This timely
book highlights some of the key challenges protein crystallography faces
and opportunities for its expansion. True to the technique’s origins, the
authors span a range of scientific disciplines. In Chapter 1 Martiel et al.
discuss in situ crystallography, a term that signals a renaissance in room
temperature crystallography, focussed on avoiding the problematic stages
of crystal handling and mounting, and is now offered in various forms
at many synchrotrons and XFELs. In the following chapter Abola et al.
develop this theme to focus on one of the major success stories of XFELs,
the determination of the structure of G-protein coupled receptors. Free
electron lasers with their brilliance and pulse time structure are beginning
to make an impact driving new techniques, as described here. This chapter
reminds us of the ultimate goal of the technique, to make a difference to
human health. Molecules that target GPCR’s account for 40% of medicines
in use but until the structural insights in 2007, the rational design of such
molecules was limited to models based on rhodopsin. The past ten years
have seen multiple new drug companies emerge using the structure of
GPCR’s to develop new medicines. Foos et al., return to the problem
addressed by Martiel of how to further optimise the use of synchrotron
radiation, this time looking at ways of optimising the collection of frag-
mentary cryo data sets; methods such as this should ultimately allow high
quality data sets to be collected rapidly as slivers and assembled together
automatically from very large numbers of microcrystals. Garman and Weik
bring the reader up to date on the decay of protein crystals upon X-ray
exposure, a problem which although diminished by cryo-crystallography
is far from eliminated. Although our knowledge is still incomplete, the
authors demonstrate how our present understanding of the problem has
led to approaches to reduce its baleful consequences and just occasionally
how it can work to our advantage. The central importance of extracting
the most accurate and meaningful data from experiments is stressed by
Vollmar and Evans who set out improvements in data processing (speed,
depth of information and accuracy) and provide a useful checklist
for collecting good quality data. The volume continues with two chapters
illustrating testing examples; Symersky et al. describe improved method-
ologies for working with low-resolution, anisotropic data and crystal
twinning while Bunker’s work shows that even low-resolution data can
transform our understanding of biology. Both of these contributions are
reminders that although technological advances make crystallography
sometimes seem almost facile, accessing interesting biology can require
painstaking work, and such work highlights weaknesses in our current
ProteinCrystallography:Faster,Smaller,Stronger ix
arsenal of techniques – it should by now be routine to be able to
refine structures against a target function derived from multiple non-
isomorphous datasets. We hope that such challenging projects will drive
the next round of methods development. Finally, Zatsepin provides an
in-depth review on recent advances in XFELs from sample delivery to data
processing. She highlights the advantages of using XFELs to study
challenging membrane proteins as well as to study enzymatic reactions
that would not be possible in conventional synchrotrons.
Finally, we should remind ourselves that the early pioneers of the field
were sharply focussed on problems of medical relevance; Hodgkin solved
penicillin,vitaminB12andinsulin,Perutzsawthestructureofhaemoglobin
as a way to explain its function and mis-function and of course lysozyme is
a natural anti-bacterial defence mechanism. Now that macromolecular
crystallography is a mature subject, capable of delivering a structure from
a well-behaved crystal in minutes, the challenge is to make the most of
the method. Hopefully this book might contribute by helping some of
the very large number of practitioners to understand the opportunities
and problems, and perhaps avoid some pitfalls.
