Biology Reference
In-Depth Information
each other. This can be considered as a 3D crystal just consisting of two
layers. However, such thin arrangements still fall into the category of 2D
crystals of interest for electron crystallography, where they can be han-
dled, rather than 3D crystals for X-ray crystallography since they do not
give rise to detectable X-ray diffraction spots. If there is a tendency for the
protein to form many layers, the electron crystallographic approach runs
into a problem since the diffraction intensity along the
z
-dimension will
be modulated by the thickness of the crystal. A tendency for crystalline
areas to stack during 2D crystallization can create an additional severe
problem. It will show up in diffraction patterns as an overlay of multiple
lattices that may be difficult to separate.
Electron Crystallography: Data Collection
and Processing
Specimen preparation and temperature
In order to collect high resolution data from 2D crystals, it is necessary to
work at low specimen temperature using cryo-electron microscopy
(cryoEM) and avoid contrast enhancing additives such as negative stain-
ing. The specimen support is normally a copper or molybdenum grid cov-
ered with a carbon layer which, in contrast to most other cryoEM
applications, is continuous in order to ensure flatness. A drop of typically
2-5
l of the 2D crystal suspension is applied to the grid. Stabilization by
freezing can proceed in two different ways. The grid can be plunged
frozen in liquid ethane as in single particle cryoEM or embedded in a low
molecular weight, non-volatile medium like glucose, tannin or trehalose
followed by freezing, which in many cases can be done in liquid nitrogen.
Most high resolution studies of 2D crystals have been made using the
embedding technique, which also allows application of a second carbon
layer to form a sandwich preparation (Gyobu
et al
., 2004). The grid is then
mounted in a conventional side entry liquid nitrogen cryo-holder and
transferred into the TEM or alternatively placed in a special cartridge if
the analysis is going to be performed with a cryo-TEM which allows spec-
imen cooling using liquid helium. In fact, most studies which have resulted
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