Biology Reference
In-Depth Information
Electron Crystallography: Initial Characterization
Whether or not 3D crystallization under certain conditions results in crys-
tals can easily be checked initially with light microscopy but the proper-
ties of crystals can only be determined by X-ray diffraction. Monitoring
2D crystallization requires a TEM. Normally, this is done after negative
staining of the specimen. This preparation method is very quick and sim-
ple and produces a long-lasting specimen which is stable under the high
vacuum conditions in the TEM column and which also tolerates a high
electron dose (for a review, see Harris and Horne (1994)). The contrast
between the staining material and the protein is high but the resolution
from crystalline material is limited to about 15 Å. Consequently, this tech-
nique does not give a definite answer to the question whether or not the
crystals give rise to high resolution information, but from the size of the
coherent crystalline areas and the sharpness of the diffraction spots in cal-
culated power spectra it is nevertheless possible to obtain a qualitative
indication regarding the size and order of the crystals.
Even if the negative stain preparation procedure is fast, checking of
the crystallinity from a large number of specimens is time consuming.
Every EM grid has to be scanned by the operator. Images from interesting
specimen areas are collected on a CCD camera attached to the TEM fol-
lowed by calculation of the corresponding power spectra online and stor-
age of representative information. Efforts have been launched in order to
speed up the whole procedure. Multi-grid specimen holders have been
constructed and automatic image acquisition systems have been tried
(Lefman
et al
., 2007). However, it is difficult to make the complete pro-
cedure fully automatic, since in particular the final evaluation step is not
simple to transfer from human judgement to computer software.
To summarize this part, it is extremely advantageous to have a TEM
facility in the vicinity of the lab where the 2D crystallization experiments
are going to be performed. It is sufficient to have a simple microscope,
and specimen preparation and instrument operation does not require a
very experienced electron microscopist. On the other hand, it is recom-
mended to have some crystallographic knowledge since already at this
Search WWH ::
Custom Search