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X-ray crystallography is
a technique in
crystallography in which the
pattern produced by the
diffraction of
X-rays through the closely
spaced lattice of
atoms in a
crystal is recorded and then
analyzed to reveal the nature of
that lattice. This generally leads
to an understanding of the
material and molecular structure
of a substance. The spacings in
the crystal lattice can be
determined using
Bragg's law. The
electrons that surround the
atoms, rather than the
atomic nuclei themselves, are
the entities which physically
interact with the incoming X-ray
photons. This technique is
widely used in
chemistry and
biochemistry to determine the
structures of an immense variety
of molecules, including inorganic
compounds,
DNA and
proteins. X-ray diffraction is
commonly carried out using single
crystals of a material, but if
these are not available,
microcrystalline powdered samples
may also be used, although this
requires different equipment,
gives less information, and is
much less straightforward.
Inorganic Structures
In inorganic chemistry, x-ray
crystallography is used to
determine lattice structures as
well as chemical formulas, bond
lengths and angles. Many
complicated inorganic and
organometallic systems have been
analyzed, such as fullerenes,
metalloporphyrins, and many other
complicated compounds. Inorganic
x-ray crystallography is commonly
known as small molecule
crystallography, as opposed to
macromolecular crystallography.
X-ray diffraction finds
frequent use in
materials science because
sample preparation is relatively
easy, and the test itself is often
rapid and non-destructive. The
vast majority of engineering
materials are crystalline, and
even those which are not yield
some useful information in
diffraction experiments.
The pattern of diffraction
peaks can be used to quickly
identify materials (thanks to the
JCPDS pattern database), and
changes in peak width or position
can be used to determine crystal
size, purity, and
texture.
An X-ray diffraction
image for the protein
myoglobin.
Biological Structures
The first
protein crystal structure was
of
sperm whale
myoglobin, as determined by
Max Perutz and
Sir John Cowdery Kendrew in
1958, which led to a
Nobel Prize in Chemistry. The
X-ray diffraction analysis of
myoglobin was originally motivated
by the observation of myoglobin
crystals in dried pools of blood
on the decks of whaling ships.
Today X-ray crystallography is
used by
pharmaceutical companies to
determine specifically how
drug lead compounds interact
with their protein targets.
Biological X-ray crystallography
is to date the most prolific
discipline within the area of
Structural biology; out of the
~35000 protein structures solved,
X-ray crystallography is
responsible for ~29000.
Nuclear Magnetic Resonance has
contributed almost 5000 and
Electron Microscopy just over
100. Other
Biophysical methods, such as
IR spectroscopy and
Powder diffraction make up the
remaining structures, according to
the Protein Data Bank (PDB).
In order to solve a protein
crystal structure, you must first
crystallise the protein. This
is becuase a single molecule in
solution has insufficent
scattering power alone. A crystal
can be considered to be an
(effectively) infinite repeating
array of our molecule of interest.
The
Laue conditions and
Bragg's_law show that
constructive
interference between
diffracted
X-rays that are
in-phase reinforce each other,
so that the
diffraction pattern becomes
detectable. The geometric
conditions where diffraction
occurs can be visuallised using
The Ewald Sphere.
However, crystallisation of
macromolecules is not trivial.
Traditional methods of
crystallising inorganic molecules
are rarely applicable as proteins
are sensitive to temperature and
high concentrations of organic
solvents. Many methods exist to
crystallise proteins, but the two
most successful methods are the
microbatch and vapour
diffusion techniques.
Concentrated solutions of the
protein are mixed with various
solutions, which typically consist
of:
- a
buffer to control the
pH of the experiment
- a
Precipitating agent, to
induce
supersaturation (typically
Poly ethylene glycols, Salts
such as Ammonium sulphate or
organic alcohols).
- other salts or additives,
such as detergents or co-factors
In either microbatch or
vapour diffusion the
solutions are allowed to
concentrate over time. In
solutions of a favourable
composition, the protein becomes
supersaturated and crystal
nuclei form, leading to
crystal growth. Typically protein
crystallographers can screen
hundreds or thousands of
conditions before a suitable
condition is found that leads to a
crystal of suitable quality. As a
rule of thumb, some useful detail
can be gained from a crystal that
diffracts with a
resolution of better than 4
Angstroms.
Many biomolecules of interest
still have not been successfully
crystallised. Imperfections in the
crystal structure, caused by
impurities or sample contamination
can prevent the acquisition of
atomic
resolution images.
Convection caused by
temperature variations within the
forming crystal can also cause
imperfections, and one of the
proposed scientific applications
of the
International Space Station is
the growth of crystals, because
convection is reduced in the free
fall environment of an orbiting
spacecraft.
Once prepared the crystals are
harvested and often cryocooled
with gaseous or liquid
nitrogen at a temperature of
around 100
kelvin or -172oC.
Liquid
helium is occasionally used
too, but it is often not necessary
to cool crystals that much (and it
also costs more). Cryocooling
crystals both reduces radiation
damage incurred during data
collection and decreases thermal
motion within the crystal, giving
rise to better
diffraction limits. Crystals
are then mounted on a
diffractometer coupled with a
machine that emits a beam of
X-rays This can either be a
rotating-anode type source or
a
Synchrotron. The X-rays are
diffracted by their interaction
with the
electrons in the crystal, and
the pattern of diffraction is
recorded on film or more recently
charge-coupled device
detectors and scanned into a
computer. Sucessive images are
recorded as a crystal is rotated
within the X-ray beam.
Data Processing
The data collected from a
diffraction experiment is a
reciprocal space
representation of the crystal
lattice. The position of each
diffraction 'spot' is governed by
the size and shape of the
unit cell, and the inherent
symmetry within the crystal.
The intensity of each diffraction
'spot' is recorded, and is
proportional to the square of the
structure factor
amplitude. The structure
factor is a
complex number containing
information relating to both the
amplitude and
phase of a
wave. In order to obtain an
interpretable electron density
map, we must first obtain
phase estimates (An electron
density map allows a
crystallographer to build a
starting model of our molecule)
This is known as the
Phase problem can be
accomplished in a variety of ways.
- Molecular Replacement - if a
structure exists of a related
protein, we can use this
structure as a search model and
use molecular replacment to
determine the orientation and
position of our molecules within
the
unit cell The phases
obtained this way can be used to
generate electron density
maps.
- Heavy Atom methods - If we
can soak high-molecular weight
atoms (not usually found in
proteins) into our crystal we
can use
direct methods or
Patterson-space methods to
determine their location and use
them to obtain initial phases.
- Ab Initio phasing -
if we have high resolution data
(better than 1.6Angstrom)
we can use direct methods to
obtain phase information.
Having obtained initial phases
we can build an initial model (our
hypothesis) and then refine
the
cartesian cordinates of atoms
and their respective B-factors
(relating to the thermal motion of
the atom) to best fit the observed
diffraction data. This generates a
new (and hopefully more accurate)
set of phases and a new electron
density map is generated. The
model is then revised and updated
by the crystallographer and a
futher round of refinement is
carried out. This continues until
the correlation between the
diffraction data and the model is
maximised.
Once the model of a molecule's
structure has been finalised, it
is often deposited in a
crystallographic database such as
the
Protein Databank or the
Cambridge Structure Database.
Many structures obtained in
private commercial ventures to
crystallise medicinally relevant
proteins, are not deposited in
public crystallographic databases.
See also
References
- Drenth J. Principles of
Protein X-Ray Crystallography.
Springer-Verlag Inc. NY: 1999,
ISBN 0387985875.
- Glusker JP, Lewis M, Rossi
M. Crystal Structure Analysis
for Chemists and Biologists.
VCH Publishers. NY:1994,
ISBN 0471185434.
- Rhodes G. Crystallography
Made Crystal Clear. Academic
Press. CA: 2000,
ISBN 0125870728.
