trehalose solution by inserting the grid deep in the solution and
slowly moving up out to the buffer surface.
7. Flip over the grid and set the tweezer and the grid on the
working bench. Mix with 2 μL of the crystal suspension by
gentle pipetting (see Note 7) (Fig. 6). Be careful not to break
the carbon film beneath the grid.
8. Let the mixture to settle for 15 s.
9. Use the pipette to remove most of the solution on the grid.
10. Use a platinum wire loop to pick up the second carbon film
from the sample buffer reservoir.
11. Gently place the second carbon film on the top of the grid.
While the wire loop touches the grid, use a filter paper to touch
the loop edge and blot away the excess solution.
12. Once the second carbon film is settled, use another piece of
filter paper to remove the remaining solution.
13. Wait for about 1 or 2 min. Directly freeze the grid specimen
into the liquid nitrogen and transfer it to the grid box for
storage.
14. Store the specimen in the liquid nitrogen dewar for later data
collection.
15. If a cryo-specimen transfer holder is used, pre-cool the cryotransfer holder and wait until the holder is stable at liquid
nitrogen temperatures. Transfer the frozen grid to the tip of
the holder and secure the grid with a clip ring. Insert the holder
to the TEM and wait until the temperature and EM vacuum are
stable for data collection.
3.2 Data Collection
For crystallographic data collection, it is important to obtain a
parallel electron beam to generate the Bragg reflections on the
diffraction pattern or image Fourier transform from the 2D crystalline specimen [35, 47]. The electron beam is required to be well
aligned to obtain high-resolution information. Different from the
diffraction obtained from a three-dimensional (3D) crystal, the
reflections of the 2D crystal pattern are the samples that are intercepted by a plane at a specific Euler angle with the 2D lattice lines
[35]. To reconstruct the 2D crystal projections into a 3D reconstruction, we collect the 2D crystal images or diffraction patterns at
different tilting angles (Fig. 6). These data will be later extracted
and merged into a set of lattice lines for 3D structure
determination.
The following procedures are written for a FEI Tecnai Polara
F30 TEM. The procedures for beam alignment can be mostly
found in the Direct Alignment panel in the vendor software. Details
can be modified for different TEMs with a field-emission gun
(FEG) source.
Electron Crystallography of Membrane Proteins
257
slowly moving up out to the buffer surface.
7. Flip over the grid and set the tweezer and the grid on the
working bench. Mix with 2 μL of the crystal suspension by
gentle pipetting (see Note 7) (Fig. 6). Be careful not to break
the carbon film beneath the grid.
8. Let the mixture to settle for 15 s.
9. Use the pipette to remove most of the solution on the grid.
10. Use a platinum wire loop to pick up the second carbon film
from the sample buffer reservoir.
11. Gently place the second carbon film on the top of the grid.
While the wire loop touches the grid, use a filter paper to touch
the loop edge and blot away the excess solution.
12. Once the second carbon film is settled, use another piece of
filter paper to remove the remaining solution.
13. Wait for about 1 or 2 min. Directly freeze the grid specimen
into the liquid nitrogen and transfer it to the grid box for
storage.
14. Store the specimen in the liquid nitrogen dewar for later data
collection.
15. If a cryo-specimen transfer holder is used, pre-cool the cryotransfer holder and wait until the holder is stable at liquid
nitrogen temperatures. Transfer the frozen grid to the tip of
the holder and secure the grid with a clip ring. Insert the holder
to the TEM and wait until the temperature and EM vacuum are
stable for data collection.
3.2 Data Collection
For crystallographic data collection, it is important to obtain a
parallel electron beam to generate the Bragg reflections on the
diffraction pattern or image Fourier transform from the 2D crystalline specimen [35, 47]. The electron beam is required to be well
aligned to obtain high-resolution information. Different from the
diffraction obtained from a three-dimensional (3D) crystal, the
reflections of the 2D crystal pattern are the samples that are intercepted by a plane at a specific Euler angle with the 2D lattice lines
[35]. To reconstruct the 2D crystal projections into a 3D reconstruction, we collect the 2D crystal images or diffraction patterns at
different tilting angles (Fig. 6). These data will be later extracted
and merged into a set of lattice lines for 3D structure
determination.
The following procedures are written for a FEI Tecnai Polara
F30 TEM. The procedures for beam alignment can be mostly
found in the Direct Alignment panel in the vendor software. Details
can be modified for different TEMs with a field-emission gun
(FEG) source.
Electron Crystallography of Membrane Proteins
257