3.3.1 Cryo-EM Grid
Preparation of Single- Or
Double-Layered 2D
Crystals
1. Two stacked pieces of Whatman #4 filter paper are placed on a
clean bench.
2. A piece of Parafilm is placed next to the filter paper on the
bench.
3. Two droplets of 150 μL of trehalose solution are pipetted onto
the Parafilm (see Note 20).
4. A grid is picked up with a pair of anticapillary forceps (see Note
21).
5. A square piece of carbon-coated mica is cut to a size slightly
larger than a grid.
6. The carbon film is floated off on the first drop (see Note 22).
7. The carbon film is picked up with the anticapillary forceps (see
Note 23).
8. The grid is touched to the surface of the second drop in order
to remove excess carbon film at the periphery of the grid.
9. The grid is rotated by 180
(the carbon side is facing down)
and the forceps are placed on the bench.
10. The side of a pipette tip is gently drawn across the surface of the
grid to remove any remaining broken pieces of carbon film.
11. A volume of 1.3–2 μL of the 2D crystal solution is pipetted
onto the grid and distributed in the solution on the grid by
pipetting 10Â.
12. The solution on the grid is incubated for 1 min.
13. The grid is rotated by 180
and placed on the two layers of filter
paper without releasing it from the forceps.
14. The grid is picked up, air-dried for 10–15 s, and hand-plunged
into liquid nitrogen, either for immediate or later use.
3.3.2 Peel-Blot
It is not uncommon for 2D crystals to “stack” into many layers,
which may impede attempts at structure determination. We have
developed a cryo-EM grid preparation (Fig. 3) technique that
mechanically separates the layers of these crystals stacks by using
the high capillary pressure from blotting cryo-EM grids prepared
by back-injection on a filter membrane of submicron pore size. This
causes the carbon film to be tightly suctioned onto the surface of
the metal grid bars, and any 2D crystals stacks positioned between
the carbon film and the grid bars adhere to both surfaces. When
these surfaces are subsequently separated by addition of buffer, the
stack layers separate, often leaving single layers adhered to the
carbon film surface that retain crystallinity and are suitable for
EM data collection (Fig. 4). This method is also capable of separating disordered lamellae, such as collapsed lipid vesicles (Fig. 5).
2D Electron Crystallography of Membrane Proteins
235
Preparation of Single- Or
Double-Layered 2D
Crystals
1. Two stacked pieces of Whatman #4 filter paper are placed on a
clean bench.
2. A piece of Parafilm is placed next to the filter paper on the
bench.
3. Two droplets of 150 μL of trehalose solution are pipetted onto
the Parafilm (see Note 20).
4. A grid is picked up with a pair of anticapillary forceps (see Note
21).
5. A square piece of carbon-coated mica is cut to a size slightly
larger than a grid.
6. The carbon film is floated off on the first drop (see Note 22).
7. The carbon film is picked up with the anticapillary forceps (see
Note 23).
8. The grid is touched to the surface of the second drop in order
to remove excess carbon film at the periphery of the grid.
9. The grid is rotated by 180
(the carbon side is facing down)
and the forceps are placed on the bench.
10. The side of a pipette tip is gently drawn across the surface of the
grid to remove any remaining broken pieces of carbon film.
11. A volume of 1.3–2 μL of the 2D crystal solution is pipetted
onto the grid and distributed in the solution on the grid by
pipetting 10Â.
12. The solution on the grid is incubated for 1 min.
13. The grid is rotated by 180
and placed on the two layers of filter
paper without releasing it from the forceps.
14. The grid is picked up, air-dried for 10–15 s, and hand-plunged
into liquid nitrogen, either for immediate or later use.
3.3.2 Peel-Blot
It is not uncommon for 2D crystals to “stack” into many layers,
which may impede attempts at structure determination. We have
developed a cryo-EM grid preparation (Fig. 3) technique that
mechanically separates the layers of these crystals stacks by using
the high capillary pressure from blotting cryo-EM grids prepared
by back-injection on a filter membrane of submicron pore size. This
causes the carbon film to be tightly suctioned onto the surface of
the metal grid bars, and any 2D crystals stacks positioned between
the carbon film and the grid bars adhere to both surfaces. When
these surfaces are subsequently separated by addition of buffer, the
stack layers separate, often leaving single layers adhered to the
carbon film surface that retain crystallinity and are suitable for
EM data collection (Fig. 4). This method is also capable of separating disordered lamellae, such as collapsed lipid vesicles (Fig. 5).
2D Electron Crystallography of Membrane Proteins
235