10. Attach fine-tip tweezers holding the EM grid and protein
microcrystal sample to the plunge freezing device.
11. Blot and vitrify the sample using the plunge freezing device (see
Note 5). Place the vitrified sample into the cryo grid box that is
under liquid nitrogen. Ensure that once the grid has been
vitrified it always remains at liquid nitrogen temperatures.
12. Repeat steps 6–11 to make multiple samples. Additional samples should be made to screen around blotting time, volume of
crystals dispensed on grid, and microcrystal treatment strategies (e.g., cryo-protectants, dilutions).
13. Once all samples have been made, store the cryo grid boxes
containing the prepared EM grids under liquid nitrogen until
loading into a cryo-TEM for analysis.
3.2 Screening
Microcrystal
Diffraction Quality
1. Follow protocols for the cryo-TEM to align the microscope in
both imaging and diffraction mode (see Note 6).
2. Load grids into a cryo-TEM equipped with a high-speed detector for diffraction data collection (see Notes 2 and 3).
3. When the samples have been loaded in the cryo-TEM, examine
the grid under low magnification (~100–200Â). This step is to
examine the overall thickness of the grid and verify presence of
protein microcrystals. It is common to observe an uneven
distribution of crystals and overall thickness. If there are no
visible regions of the grid because grid is too thick everywhere,
or if there are no crystals visible on the grid, either load a new
grid or repeat the sample preparation steps to prepare new
samples with different blotting or sample handling conditions.
4. Note areas of the grid which are promising (i.e., thin enough
for the beam to penetrate and holding crystals). Move the stage
to one of these grid squares and increase the magnification to a
medium level (approximately 500–1000Â). The field of view
should be approximately one grid square (see Fig. 1).
5. Locate promising crystals on the grid square. If no crystals are
visible when zooming in after step 4, return to step 4, and
move to a new grid square.
6. When a promising crystal is identified (well-defined edges and
well-separated from other crystals or the grid bars) on the grid
square, move the crystal to the center of the detector. The
eucentric height should be accurately set so that the crystal
does not move as the stage is rotated.
7. Insert the selected area aperture.
8. Insert the beam stop, unless the detector equipped on the cryoTEM does not require the use of a beam stop.
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291
microcrystal sample to the plunge freezing device.
11. Blot and vitrify the sample using the plunge freezing device (see
Note 5). Place the vitrified sample into the cryo grid box that is
under liquid nitrogen. Ensure that once the grid has been
vitrified it always remains at liquid nitrogen temperatures.
12. Repeat steps 6–11 to make multiple samples. Additional samples should be made to screen around blotting time, volume of
crystals dispensed on grid, and microcrystal treatment strategies (e.g., cryo-protectants, dilutions).
13. Once all samples have been made, store the cryo grid boxes
containing the prepared EM grids under liquid nitrogen until
loading into a cryo-TEM for analysis.
3.2 Screening
Microcrystal
Diffraction Quality
1. Follow protocols for the cryo-TEM to align the microscope in
both imaging and diffraction mode (see Note 6).
2. Load grids into a cryo-TEM equipped with a high-speed detector for diffraction data collection (see Notes 2 and 3).
3. When the samples have been loaded in the cryo-TEM, examine
the grid under low magnification (~100–200Â). This step is to
examine the overall thickness of the grid and verify presence of
protein microcrystals. It is common to observe an uneven
distribution of crystals and overall thickness. If there are no
visible regions of the grid because grid is too thick everywhere,
or if there are no crystals visible on the grid, either load a new
grid or repeat the sample preparation steps to prepare new
samples with different blotting or sample handling conditions.
4. Note areas of the grid which are promising (i.e., thin enough
for the beam to penetrate and holding crystals). Move the stage
to one of these grid squares and increase the magnification to a
medium level (approximately 500–1000Â). The field of view
should be approximately one grid square (see Fig. 1).
5. Locate promising crystals on the grid square. If no crystals are
visible when zooming in after step 4, return to step 4, and
move to a new grid square.
6. When a promising crystal is identified (well-defined edges and
well-separated from other crystals or the grid bars) on the grid
square, move the crystal to the center of the detector. The
eucentric height should be accurately set so that the crystal
does not move as the stage is rotated.
7. Insert the selected area aperture.
8. Insert the beam stop, unless the detector equipped on the cryoTEM does not require the use of a beam stop.
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