TEM images reveal details of the crystal lattices that prove to be
accurate qualitative indicators of the potential diffraction of the
crystal. In general, the detection by negative stain TEM methods
of well-ordered lattices with third order Bragg spots was a good
predictor of X-ray diffraction, while samples with disorganized
lattices yielded no diffraction [12]. Additionally, we show that
TEM can play an important role to identify crystal pathologies
that contribute to poor X-ray diffraction data [11]. Among them
are: (a) crystal lattice defects; (b) anisotropic diffraction; and
(c) crystal “polluting” by heavy protein aggregates and microcrystal
nuclei. Detection of lattice defects in some crystals could point to
the presence of samples containing protein contaminants, aggregates or partially proteolyzed protein as well as discrepancies in the
stoichiometry of the sample. Negative stain TEM analysis was able
to identify crystals that possess anisotropic diffraction. In these
cases, performing steps to improve crystal contacts, such as altering
or adding reagents to the crystallization conditions or modification
of the protein itself, may be advisable. It is important to note that
this information cannot be observed with other techniques before
performing X-ray diffraction experiments. The use of TEM has also
enabled qualitative estimation of crystal solvent content and
allowed the study of lattice dehydration on crystal diffraction
[11]. This application was particularly noteworthy since (a) crystal
dehydration protocols have proven very useful in the improvement
of X-ray diffraction [13–15], and (b) negative staining with uranyl
acetate provides very high contrast between solvent channels and
biological macromolecules. Overall, information obtained by TEM
experiments could provide critical advice to the experimenter about
which crystallization conditions to be pursued and would also allow
monitoring of crystal optimization protocols.
2 Materials
2.1 Microscopy
1. Brightfield and light microscope.
2. Ultraviolet (UV) microscope.
3. Electron microscope (e.g., FEI TECNAI T12 operating at
120 kV with a single-tilt specimen holder and 2 k  2 k
Gatan UltraScan 1000 CCD camera).
2.2 Negative
Staining Components
1. 2% (w/v) uranyl acetate in 15 mL plastic Falcon tube, covered
in aluminum foil.
2. 0.22 μm sterile filter.
3. Carbon-coated CF400-CU grids.
4. Glow discharge unit.
5. Filter paper.
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Simon Weiss et al.
accurate qualitative indicators of the potential diffraction of the
crystal. In general, the detection by negative stain TEM methods
of well-ordered lattices with third order Bragg spots was a good
predictor of X-ray diffraction, while samples with disorganized
lattices yielded no diffraction [12]. Additionally, we show that
TEM can play an important role to identify crystal pathologies
that contribute to poor X-ray diffraction data [11]. Among them
are: (a) crystal lattice defects; (b) anisotropic diffraction; and
(c) crystal “polluting” by heavy protein aggregates and microcrystal
nuclei. Detection of lattice defects in some crystals could point to
the presence of samples containing protein contaminants, aggregates or partially proteolyzed protein as well as discrepancies in the
stoichiometry of the sample. Negative stain TEM analysis was able
to identify crystals that possess anisotropic diffraction. In these
cases, performing steps to improve crystal contacts, such as altering
or adding reagents to the crystallization conditions or modification
of the protein itself, may be advisable. It is important to note that
this information cannot be observed with other techniques before
performing X-ray diffraction experiments. The use of TEM has also
enabled qualitative estimation of crystal solvent content and
allowed the study of lattice dehydration on crystal diffraction
[11]. This application was particularly noteworthy since (a) crystal
dehydration protocols have proven very useful in the improvement
of X-ray diffraction [13–15], and (b) negative staining with uranyl
acetate provides very high contrast between solvent channels and
biological macromolecules. Overall, information obtained by TEM
experiments could provide critical advice to the experimenter about
which crystallization conditions to be pursued and would also allow
monitoring of crystal optimization protocols.
2 Materials
2.1 Microscopy
1. Brightfield and light microscope.
2. Ultraviolet (UV) microscope.
3. Electron microscope (e.g., FEI TECNAI T12 operating at
120 kV with a single-tilt specimen holder and 2 k  2 k
Gatan UltraScan 1000 CCD camera).
2.2 Negative
Staining Components
1. 2% (w/v) uranyl acetate in 15 mL plastic Falcon tube, covered
in aluminum foil.
2. 0.22 μm sterile filter.
3. Carbon-coated CF400-CU grids.
4. Glow discharge unit.
5. Filter paper.
300
Simon Weiss et al.