11. Cryo grid box.
12. Cryo-TEM (see Note 2).
13. High-speed detector for the collection of electron diffraction
data (see Note 3).
3 Methods
Here, we will present the generic workflow for MicroED sample
preparation and data collection when working with protein crystals.
The procedures start after the identification of protein microcrystals [22, 31–33] and ends with the collection of continuous rotation MicroED data sets that can be processed and used for structure
determination [22]. It is recommended that first time users of
MicroED attempt these protocols with known standards in order
to become familiar with the process. Suggested protein standards
are lysozyme and proteinase K and there are several publications
where these samples have been analyzed by MicroED [6, 7, 9, 34].
3.1 Sample
Preparation
1. Use clean tweezers to place carbon-coated EM grids onto a
clean glass microscope slide.
2. Use a glow discharge unit and follow the manufacturer’s protocol to clean the surface of the carbon.
3. Use tweezers to flip the grids over on the glass slide and repeat
step 2 to treat the opposite surface of the grid.
4. Assemble the components of the plunge freezing coolant container according to the specific manufacturer’s instructions. Fill
container with liquid nitrogen and allow the temperature to
stabilize. Add additional liquid nitrogen as needed.
5. Fill the reservoir where the grid will be plunged with liquid
ethane according to manufacturer’s instructions.
6. Use the locking fine-tip tweezers for plunge freezing apparatus
to pick up an EM grid prepared following step 3 and place
tweezers on the lab bench.
7. Use a pipette to aspirate 2–3 μL of solution containing microcrystals (see Note 4). Dispense solution onto the EM grid.
8. Allow crystal solution to rest on the surface of the grid for 30 s
to 1 min.
9. In some cases, microcrystal solution is applied to both sides. If
crystals will only be deposited on one side of the grid, skip this
step. To deposit crystals on the other side of the grid as well,
flip over the tweezers holding the EM grid that has just had
microcrystal solution applied to it, and repeat steps 7 and 8 to
deposit on to the other side of the grid.
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Guanhong Bu and Brent L. Nannenga
12. Cryo-TEM (see Note 2).
13. High-speed detector for the collection of electron diffraction
data (see Note 3).
3 Methods
Here, we will present the generic workflow for MicroED sample
preparation and data collection when working with protein crystals.
The procedures start after the identification of protein microcrystals [22, 31–33] and ends with the collection of continuous rotation MicroED data sets that can be processed and used for structure
determination [22]. It is recommended that first time users of
MicroED attempt these protocols with known standards in order
to become familiar with the process. Suggested protein standards
are lysozyme and proteinase K and there are several publications
where these samples have been analyzed by MicroED [6, 7, 9, 34].
3.1 Sample
Preparation
1. Use clean tweezers to place carbon-coated EM grids onto a
clean glass microscope slide.
2. Use a glow discharge unit and follow the manufacturer’s protocol to clean the surface of the carbon.
3. Use tweezers to flip the grids over on the glass slide and repeat
step 2 to treat the opposite surface of the grid.
4. Assemble the components of the plunge freezing coolant container according to the specific manufacturer’s instructions. Fill
container with liquid nitrogen and allow the temperature to
stabilize. Add additional liquid nitrogen as needed.
5. Fill the reservoir where the grid will be plunged with liquid
ethane according to manufacturer’s instructions.
6. Use the locking fine-tip tweezers for plunge freezing apparatus
to pick up an EM grid prepared following step 3 and place
tweezers on the lab bench.
7. Use a pipette to aspirate 2–3 μL of solution containing microcrystals (see Note 4). Dispense solution onto the EM grid.
8. Allow crystal solution to rest on the surface of the grid for 30 s
to 1 min.
9. In some cases, microcrystal solution is applied to both sides. If
crystals will only be deposited on one side of the grid, skip this
step. To deposit crystals on the other side of the grid as well,
flip over the tweezers holding the EM grid that has just had
microcrystal solution applied to it, and repeat steps 7 and 8 to
deposit on to the other side of the grid.
290
Guanhong Bu and Brent L. Nannenga