6. Flip the grid upside down and set the tweezer and the grid on
the working bench (Fig. 5). A small volume (less than about
1 μL) of the embedding buffer should be maintained on the
top of the grid.
7. Apply 2 μL of the crystal suspension on the grid and gently mix
the crystal suspension with the remaining buffer on the grid by
pipetting (see Note 7).
8. Wait about 10 s and then remove the solution from the grid
using a pipette.
9. Use the filter paper to remove the remaining solution from the
grid. With glucose embedding, the grid specimen does not
need to freeze into the liquid nitrogen before transferring to
the EM stage.
10. If a side-entry cryo-specimen transfer holder is used, secure the
grid on the tip of the holder with a clip ring at room temperatures and insert the holder into the TEM. Wait until the TEM
vacuum is stable.
11. When the holder is in the TEM column, add liquid nitrogen to
the dewar and cool down the holder.
12. Closely monitor the temperature of the cryo-specimen transfer
holder. When the tip reaches the liquid nitrogen temperature,
wait about 10–15 min until the liquid nitrogen stops boiling.
Then start the procedure for data collection.
3.1.3 Carbon-Sandwich
Method
The carbon-sandwich method has the advantages of the reduction
of the beam-induced charging and the better protection of the
crystal sample with two layers of carbon films [39]. The following
protocol is for the carbon-sandwich method with trehalose embedding for sample preparation.
1. Optimize the crystal concentration using negative-stain EM.
2. If a recycled molybdenum grid is used, clean the grid with
plasma cleaning before use to remove the residuals on the
grid (see Note 8).
3. Prepare two reservoirs, one with trehalose solution and the
other with the mother buffer (see Note 9).
4. Cut a piece of carbon film from the mica sheet with a size
similar or slightly smaller than the size of the molybdenum
grid. Float a piece of carbon film on the surface of trehalose
solution. The floated carbon film should be flat and intact.
5. Float another piece of carbon film on the surface of the sample
mother buffer. The size of the carbon film should be smaller
than that of the first piece (see Note 10).
6. Use an anti-capillary self-closing tweezer to pick up an EM
grid. Pick up the carbon film that is on the surface of the
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the working bench (Fig. 5). A small volume (less than about
1 μL) of the embedding buffer should be maintained on the
top of the grid.
7. Apply 2 μL of the crystal suspension on the grid and gently mix
the crystal suspension with the remaining buffer on the grid by
pipetting (see Note 7).
8. Wait about 10 s and then remove the solution from the grid
using a pipette.
9. Use the filter paper to remove the remaining solution from the
grid. With glucose embedding, the grid specimen does not
need to freeze into the liquid nitrogen before transferring to
the EM stage.
10. If a side-entry cryo-specimen transfer holder is used, secure the
grid on the tip of the holder with a clip ring at room temperatures and insert the holder into the TEM. Wait until the TEM
vacuum is stable.
11. When the holder is in the TEM column, add liquid nitrogen to
the dewar and cool down the holder.
12. Closely monitor the temperature of the cryo-specimen transfer
holder. When the tip reaches the liquid nitrogen temperature,
wait about 10–15 min until the liquid nitrogen stops boiling.
Then start the procedure for data collection.
3.1.3 Carbon-Sandwich
Method
The carbon-sandwich method has the advantages of the reduction
of the beam-induced charging and the better protection of the
crystal sample with two layers of carbon films [39]. The following
protocol is for the carbon-sandwich method with trehalose embedding for sample preparation.
1. Optimize the crystal concentration using negative-stain EM.
2. If a recycled molybdenum grid is used, clean the grid with
plasma cleaning before use to remove the residuals on the
grid (see Note 8).
3. Prepare two reservoirs, one with trehalose solution and the
other with the mother buffer (see Note 9).
4. Cut a piece of carbon film from the mica sheet with a size
similar or slightly smaller than the size of the molybdenum
grid. Float a piece of carbon film on the surface of trehalose
solution. The floated carbon film should be flat and intact.
5. Float another piece of carbon film on the surface of the sample
mother buffer. The size of the carbon film should be smaller
than that of the first piece (see Note 10).
6. Use an anti-capillary self-closing tweezer to pick up an EM
grid. Pick up the carbon film that is on the surface of the
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Ka-Yi Chan et al.