Biology Reference
In-Depth Information
In this chapter some basic principles for obtaining 2D crystals of both
membrane-bound and soluble proteins will be described together with
their analysis and properties.
Why Two-Dimensional Crystals?
In order to observe scattering of X-ray frequency range photons giving
rise to diffraction spots with sufficiently high signal-to-noise ratio at a
resolution which allows building of atomic models of proteins, it is
required that the molecules are arranged into well-ordered crystals in three-
dimensions (3D). For some specimens this may be difficult to achieve.
Factors that may hamper 3D crystallization include heterogeneity,
hydrophobicity and tendency of aggregation at high concentration. Even
if the protein can form 3D crystals resulting in a native data set, it may be
difficult to produce heavy atom data derivatives or selenomethionine
incorporation if that is needed for phasing or to study structure-function
relationships related to ligand binding which may require an environment
resembling the natural state.
Since electrons interact more strongly with matter than X-rays do,
electron diffraction patterns and images can be observed in a transmission
electron microscope (TEM) from protein molecules periodically arranged
in a single layer. This arrangement is called a two-dimensional (2D) crys-
tal. Thus, 2D crystals combined with electron crystallography presents an
interesting alternative methodological approach for systems where 3D
crystallization is likely to fail. Unfortunately, it is difficult to predict the
outcome of crystallization trials and consequently there is no strict rule-
of-thumb for selecting one or the other of the two techniques. Since struc-
ture solution by X-ray diffraction has become very efficient and well
established it is not unusual to try to circumvent initial problems by mod-
ifications of the protein such as introducing mutations or performing trun-
cations. However, with improvements in electron crystallography both
with regard to data collection and data processing it may be equally inter-
esting to set up extensive 2D crystallization experiments and to solve the
structure using the TEM.
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