Biochemistry
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A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline
Chapters
Summary June 17th, 2021
The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.
Transcript
Standard sample preparation make it difficult to achieve a good signal-to-noise during x-ray diffraction experiments of microcrystals. This protocol aims to reduce the sources of background formed crystals in a controlled manner. The use of punch freezing robotics and cryoTEM grids provides a robust platform for manipulating the microcrystals repeatedly, reducing the surrounding liquid volume and providing rapid vitrification of the sample.
The dexterity needed to handle grids is similar to that needed for traditional crystal harvesting. Another important factor is the determination of initial blotting parameters using the crystallization solution, which is key to reducing sample usage. To begin, set up and cool the automated plunge freezer according to the manufacturer's instructions.
Just before use, glow discharge the cryoTEM grids for 25 seconds with a 15 milliampere current and 0.39 millibar pressure, then keep the glow discharge grids in a covered Petri dish. Set the relative humidity of the sample chamber to 90%and the blotting time to five seconds. Ensure that the plunge freezer is set to automatically plunge the sample after blotting is complete.
Open the seal of the crystallization well and reservoir. Quickly add two to five microliters of the reservoir solution in the crystal drop to maintain the volume of the drop. Transfer 10 microliters of the reservoir solution to a 0.5 milliliter tube for later use and reseal the well to prevent the crystallization drop from drying.
Use the plunge freezing forceps to pick a single glow discharge grid and load the grid in the instrument with the carbon side facing away from the blotting arm. Rotate the forceps holding the grid so that the carbon side faces the blotting arm. Use a 2.5 microliter pipette to apply two microliters of reservoir solution to the non-support side of the cryoTEM grid.
Rotate the grid with the carbon side facing away from the blotting arm and carefully apply the reservoir liquid to the carbon film support side of the grid. Initiate the blotting process and observe the liquid drawn from the carbon surface until the wave of popping across the surface of the grid is visible. If the wave of popping is not visible, increase the blotting time by one to two seconds before the blotting arm retracts from the grid.
Place the crystallization plate under the light microscope and position the target well within the field of view. Place a fresh glow discharge grid in the plunge freezer and apply the reservoir liquid to the non-support side of the grid as previously demonstrated. Rotate the grid with the carbon film support side facing the sample port of the plunge freezer.
Peel the temporary seal from the crystallization plate and use the pipette set at two microliters to gently aspirate the crystallization drop repeatedly. Transfer two microliters of the aspirated microcrystal slurry to the plunge freezer and apply all the sample to the carbon side of the cryoTEM grid. Initiate the blotting and observe for the wave of popping, then immediately initiate the plunge freezing.
Quickly transfer the grid from the liquid ethane to the grid box immersed in liquid nitrogen. After blotting and plunge freezing the grid, retract the plunged grid from the liquid ethane by resetting the plunge freezer. Remove the forceps holding the grid from the plunge freezer and place the grid under the light microscope.
Adjust the fine focus and assess the density of the crystals across the grid. After loading the cryoTEM grid in the SEM, align the sample and turn on the electron beam. Initially assess the whole grid at a 45X magnification and record the image, then increase the magnification for a closer inspection of individual grid squares until the individual crystals are clearly observed.
Move around the grid and capture the still images, ensuring that the grids are flat and the carbon support film is largely intact. Ensure that there are numerous single crystals with a narrow halo of vitrified liquid surrounding the crystal and the holes are visible in the carbon support film. While observing and capturing the images of the grid, make sure that the large regions of vitrified liquid are absent, hexagonal ice or surface ice is not scattered across the grid, and that the crystals are not overlapping and evenly distributed across the support film.
In a large foam dewar, cool the required number of VMXm sample holders loaded in the sample cartridge. Add liquid nitrogen above the sample position in the sample loader. Swiftly transfer the grid box containing microcrystal-loaded grids to the grid box recess on the sample loader and slightly unscrew the lid to keep the lid loose and rotatable.
Lift the grid from the grid box and rotate the grid to lay the grid flat on the sample holder. Swiftly place the pre-cooled circlip tool over the grid in the grid opening and press the button to install the circlip. Add liquid nitrogen approximately 1.5 centimeters above the sample holder.
Use the VMXm sample forceps to carefully lift the loaded sample holder and place it back into the sample cartridge. Replace the lid on the cartridge, ensuring that the pin on the top of the cartridge engages with the hole in the lid. The scanning electron micrographs of microcrystals prepared on cryoTEM grids showed minimum background scatter.
The grid was free from excess liquid and a narrow halo of liquid was observed surrounding the crystals. The polyhedral crystals were observed individually as well as in clumps. Slightly larger insulin crystals also showed some clumping along with isolated crystals.
Very large microcrystals were also successfully mounted on cryoTEM grids. The holes in the carbon support film were clearly visible, indicating strong blotting. Many samples required further optimization because of variation in blotting time and concentration of the microcrystals.
The grids overloaded with crystals reduce the blotting efficiency and multiple lattices were recorded in a single diffraction image. Short blotting time for highly viscous crystallization solutions can result in an overly wet sample. For a lower viscosity crystallization solution, a short blotting time results in stealing of microcrystals on one side of the grids.
Optimum sample preparation makes it possible to exploit the full capabilities of VMXm to collect high-quality x-ray diffraction data at the highest possible resolution with a high signal-to-noise ratio. Determining initial blotting of a crystallization solution is key to using minimal sample. If the sample is very limited, skip the density assessment.
Ultimately, it's better to have a diluted sample. These samples are now ready for x-ray diffraction experiments at the VMXm Beamline, microcrystal electron diffraction, or focused ion beam milling prior to micro ED.
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