Biology Reference
In-Depth Information
unbending lattices in images, correcting for the contrast transfer function,
merging data in 2D and 3D, lattice line adaption and finally ending up
with a complete set of Fourier components consisting of the Miller indices,
amplitude values, standard deviations of the amplitudes, phases and fig-
ures-of-merit of the phases. Alternative program systems have been devel-
oped which may or may not use the MRC programs in the background at
the computational level (Gipson
et al
., 2007; Philippsen
et al
., 2007). The
complete 3D dataset can subsequently be transferred to a conventional
X-ray crystallography package like CCP4 for further processing, map
calculation, model building and refinement.
2D or 3D Crystallization?
For the purpose of structural studies, crystallization in general is just a
means of improving signal-to-noise ratio in recorded diffraction/image
data. Ideally, the crystalline arrangement should as closely as possible
resemble the natural
in situ
environment and not induce irrelevant struc-
tural changes of the studied macromolecule. Furthermore, the system
should preserve activity and allow induction of conformational changes
that may be directly coupled to the function.
For soluble proteins, 2D crystallization generates an artificial system
except in those special cases when proteins have a natural tendency to
interact with membranes. Moreover, even if production of large well-
ordered 2D crystals on lipid monolayers has been demonstrated, there is
no example yet of a structural study that has resulted in an atomic model.
For membrane proteins, however, 2D crystals resemble the natural
environment and there are even examples of naturally occurring crys-
talline sheets of very densely packed proteins. Many membrane proteins
expose active sites to either side of the lipid bilayer and these areas are
accessible also in 2D crystals, implying that the effects of ligand binding
can be studied. Furthermore, conformational states can be frozen and
studied separately (Unwin, 2003). Membrane bound enzymes may also
have hydrophobic substrates that are interacting with an active site in the
lipophilic region also accessible in 2D crystals. Furthermore, two or more
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