Protein crystal structure determination.
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Protein crystal structure determination.
The determination of a novel protein structure by X-ray crystallographic analysis involves the following
steps. i) Protein purification and crystallisation of the native protein, ii) measurement of native
diffraction data, iii) obtaining heavy atom derivatives, iv) measurement and analysis of derivative data,
v) calculation of phases, vi) map interpretation and model building and vii) model refinement.
Crystallisation is often the rate limiting step in protein crystallography. Several methods of
crystallisation are now well established but application of these methods is still very much trial and
error. Crystallisation of a newly isolated protein can take weeks, months or years.
A prerequisite of the solution of the majority of protein crystal structures is to find an isomorphous
heavy atom derivative of the native protein. Isomorphous, in this sense, means that the derivative protein
crystal structure should be identical to the native protein structure except for the presence of one or more
heavy atoms bound to protein molecules, i.e. the lattice, space group, cell dimensions and position and
conformation of the protein molecule within the unit cell should be preserved. The most common
method of heavy atom inclusion is to soak the native protein crystals in a solution containing a heavy
atom compound. Other more recent methods involve the production of protein with modified amino acid
residues, e.g. methionine containing proteins can be engineered where selenium replaces the sulphur of
the methionine [50].
Once suitable native and derivative protein crystals become available, X-ray diffraction data are
collected. One of several procedures may be adopted for data collection. Most single crystal diffraction
data are measured using monochromatic X-rays from either a sealed tube generator, a rotating anode
generator or from a synchrotron source. The availability of synchrotron radiation has led to the
application of the less widely used Laue method of data collection to protein crystallography. It utilises
the non-characteristic polychromatic radiation produced by a synchrotron. A detailed description of the
Laue method may be found in [47].
Several data collection geometries may be used along with monochromatic radiation. A conventional
diffractometer measures each reflection individually with a scintillation counter. A goniometer rotates
the crystal so as to satisfy the Bragg condition for each reflection individually while the detector
simultaneously records the diffracted X-ray intensity. Diffractometers are still widely used for small
molecule work but have recently been superseded by area detectors which are able to measure
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Protein crystal structure determination.
equivalent quantities of data in a much shorter time; an important factor when crystal samples are highly
sensitive to the dosage of X-rays delivered during the experiment. This is particularly relevant for
biological samples.
There are two area detector geometries which are at present widely used - rotation geometry and
Weissenberg geometry. In both methods the Bragg condition is satisfied by rotating the sample crystal
about a fixed axis. A series of diffraction images are measured whereby each image records all
reflections satisfying the Bragg condition as the crystal is rotated through a specified angle . A
limited value of is necessary so as to avoid overlap of diffracted reflections on an image.
The essential difference is that a Weissenberg camera couples the sample rotation with a translation of
the area detector. This helps avoid the problem of overlapping spots and uses the available space on the
area detector more efficiently and can further reduce the overall time for data collection. The use of
larger angular rotation ranges for each diffraction image in the Weissenberg geometry implies the
accumulation of more background radiation per image, however large sample to detector distances, ,
are also typically used which reduce the level of recorded background relative to the intensity of
diffracted X-rays by a factor of .
In a diffraction experiment one can only measure the intensities and diffraction angles of the diffracted
beams. All information about the phases of the diffracted X-rays is lost. This phase information along
with the amplitudes of the diffracted X-rays is essential for the solution of crystal structures and must be
recovered.
There are four approaches which may be taken in recovering phase information in a diffraction
experiment. The heavy atom method makes the assumption that if a significant contribution of the
scattering from a structure is made by a heavy atom then the phases of the diffracted X-rays will be close
to those phases which would be observed were only the heavy atoms present. The problem is thus
reduced to finding the positions of the heavy atoms within the structure. This approach is in general not
applicable to proteins since the heavy atom contribution to scatteing is small with respect to the protein.
The direct methods approach can make estimates about the reflection phases using assumptions about
the internal structure of the crystal. Direct methods are routinely used for the solution of small structures
and have only recently been applied to the solution of small proteins containing about 50 amino acid
residues [102].
If a related or similar protein structure is already known then the method of Molecular Replacement
(MR) [98] may be used. The idea is to find the rotation and translation which position the model
structure in the unit cell so as to give the highest correlation between experimental diffraction
measurements and those calculated from the model. This method relies on the existence of a known
related structure and is therefore likely to become more and more applicable as the number of solved
protein structures increases in the future.
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Protein crystal structure determination.
The third and most prominent of the solutions to the phase problem in macromolecular crystallography
is isomorphous replacement and related methods. In these methods phase information is retrieved by
making isomorphous structural modifications to the native protein, usually by including a heavy atom or
changing the scattering strength of a heavy atom already present and then measuring the diffraction
amplitudes for the native protein and each of the modified cases. If the position of the additional heavy
atom or the change in its scattering strength is known then the phase of each diffracted X-ray can be
determined by solving a set of simultaneous phase equations. Methods which use such a strategy are
Single Isomorphous Replacement (SIR), Multiple Isomorphous Replacement (MIR), Single
Isomorphous Replacement with Anomalous Scattering (SIRAS) and within the last 15 years, the
Multiple wavelength Anomalous Diffraction method (MAD).
With an experimental set of phases obtained from either direct methods or Isomorphous Replacement
related methods one can calculate a 3-dimensional electron density map of the protein structure. This is
not always readily interpretable as a single polypeptide chain and methods are usually employed to
improve the density map using knowledge about the common characteristics of protein crystals. e.g. they
nearly always contain between and solvent, are made of individual amino acid residues with
known structures and have predictable secondary structures e.g. -helix or -pleated sheet. Density
interpretation and model building have been semi-automated with the recent development of powerful
graphical computer hardware and software aimed specifically at macromolecular modelling, e.g. the
program O [61]. After and during the main phase of model building, refinement of the model is carried
out against the experimentally measured intensities. This stage may include the addition of ordered
solvent molecules and if very high resolution X-ray data are available even the addition of hydrogen
atoms, although this is rare for macromolecular structures.
next up previous contents index
Next: Multiple wavelength methods. Up: Protein Crystallography. Previous: Protein structure.
Gwyndaf Evans
Fri Oct 7 15:42:16 MET 1994
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