Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation

There have been many recent technological advances in cryo-EM, including advancements in the microscope hardware itself that improve the overall quality of the images, and also developments in software that are used to process the cryo-EM images that can start to deal with the large amounts of heterogeneity present in many of the samples. The sample preparation remains one of the largest bottlenecks in cryo-EM. The sample quantity may be limiting, and proteins tend to adsorb to the hydrophobic air-water interface during vitrification.

This can lead to sample denaturation, the breaking apart of larger protein complexes, and preferred particle orientations that are present in the cryo-EM images. These grids take advantage of the high-affinity interaction between streptavidin and biotin to tether biotinylated samples and protect them from the hydrophobic air-water interface. We can work with very low sample amounts, and random biotinylation of samples offers a strategy to overcome preferential orientation issues that may be observed when using other support layers.

To begin, clean the bench area with 70%ethanol, then rinse a five-microliter glass syringe several times with chloroform. Wash the carbon-evaporated grids with 100%ethanol just before use. Dry the grids on a clean filter paper.

In the meantime, wash each anti-capillary tweezer with 100%chloroform followed by 100%ethanol. Prepare three 50-microliter drops of crystallization buffer on the clean side of a Parafilm for each grid to be prepared. Fill the lid with the crystallization buffer and wipe the surface of a 35-millimeter uncoated petri dish with lens paper.

Sprinkle enough scientific-grade talcum powder around the perimeter of the petri dish. Dip a 200-microliter pipette tip into the castor oil to pick up a medium-sized drop. Touch the drop onto the surface of the buffer in the petri dish.

Rinse the five-microliter glass syringe twice with the dissolved lipid before taking up an aliquot. Then, gently touch the hanging 0.5-microliter lipid droplet to the surface of the castor oil, forming a lipid monolayer at the center. Prepare a plate with several sequential drops of liquid.

Pick up a grid with an anti-capillary tweezer so that the straight arm of the tweezer and the carbon-evaporated side of the grid face the monolayer. To transfer the monolayer, touch the carbon-evaporated side of the grid to the monolayer for one to two seconds. Touch the spherical cap of the buffer sequentially to the three 50-microliter drops of crystallization buffer placed on the Parafilm.

Add four microliters of streptavidin into the remaining spherical cap of the buffer on the grid. Incubate the grid at room temperature for two hours in a humidity chamber. Prepare a 300-microliter drop of rinse buffer on the clean side of a Parafilm.

Place the grid on the 300-microliter drop of rinse buffer to wash the unbound streptavidin. To pick up the grid, stick the kinked arm of a dry anti-capillary tweezer into the drop. Blot the grid with filter paper, place it with the gold side down, and dry it for 15 to 20 minutes.

Once dried, flip the grid over so the gold side faces up. Place the grids into the carbon evaporator to add a thin layer of carbon onto the gold side of the grid. The micrograph of a biotinylated sample frozen with streptavidin affinity grids showed a continuous grid pattern in the background.

The diffraction pattern in the fast Fourier transform further confirmed a successful lattice formation. After cryo-EM data collection, the signal contributed by the streptavidin lattice could be computationally masked to produce a subtracted micrograph. The fast Fourier transform results showed that the diffraction pattern for streptavidin was successfully removed.