A step-by-step protocol for fabricating streptavidin affinity grids is provided for use in structural studies of challenging macromolecular samples by cryo-electron microscopy.
Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, including sample denaturation and preferential orientations that can occur due to the air-water interface. Streptavidin affinity grids, however, are currently utilized by few cryo-EM labs because they are not commercially available and require a careful fabrication process. Two-dimensional streptavidin crystals are grown onto a biotinylated lipid monolayer that is applied directly to standard holey-carbon cryo-EM grids. The high-affinity interaction between streptavidin and biotin allows for the subsequent binding of biotinylated samples that are protected from the air-water interface during cryo-EM sample preparation. Additionally, these grids provide a strategy for concentrating samples available in limited quantities and purifying protein complexes of interest directly on the grids. Here, a step-by-step, optimized protocol is provided for the robust fabrication of streptavidin affinity grids for use in cryo-EM and negative-stain experiments. Additionally, a trouble-shooting guide is included for commonly experienced challenges to make the use of streptavidin affinity grids more accessible to the larger cryo-EM community.
Electron cryo-microscopy (cryo-EM) has revolutionized the field of structural biology by enabling macromolecular structure determination of large, flexible, and heterogeneous samples that were previously inaccessible by X-ray crystallography or nuclear magnetic resonance1. This method works by flash-freezing macromolecules in solution to create a thin layer of vitreous ice that can be subsequently imaged using an electron microscope. In recent years, significant advances in both microscope hardware and image processing software have further expanded the types of samples suitable for high-resolution structure determination by cryo-EM.
Nevertheless, the preparation of thin, vitrified samples remains one of the most critical steps in macromolecular structure determination by cryo-EM. Biological samples are often dynamic, fragile, prone to denaturation, and sometimes are only available in small quantities for cryo-EM studies. During the blotting process, these particles interact with the hydrophobic air-water interface, which can result in particle-preferred orientations, disassembly of fragile complexes, partial or complete sample denaturation, and aggregation2,3,4. The use of detergents or other surfactants, chemical cross-linking, and adsorption of samples to support layers are common strategies to preserve biological samples during the freezing process. Support layers such as graphene oxide5,6,7 or amorphous carbon8 also function to concentrate particles on the grid by adsorption when the sample is available in limited quantities. However, these methods are not general or reliable, and optimization of grid preparation can be extremely time-consuming or fail altogether.
Streptavidin affinity grids9,10 were developed to overcome these shortcomings and to provide a mild and generally applicable method to sequester the complex of interest and protect it from the air-water interface. These grids utilize a two-dimensional (2D) streptavidin crystal lattice grown on a monolayer of biotinylated lipids on the grid. After samples are themselves biotinylated (often sparsely and randomly, meaning one biotin per complex on average), they can be applied to the streptavidin-coated grid. Because sample adsorption relies on the extremely high affinity between streptavidin and biotin, sample concentrations as low as 10 nM can be used with these grids. Commercially available biotinylation kits for proteins and biotinylated primers for DNA-containing complexes make it relatively easy to attach the necessary biotin moieties to most samples of interest. In addition to concentrating the sample and keeping it away from the damaging air-water interface during blotting, the random biotinylation of one or just a few lysine residues can significantly improve the range of orientations of the molecule of interest on the cryo-EM grid, as demonstrated in a number of studies11. While the signal from the underlying streptavidin crystal is present in the raw images, data processing schemes involving Fourier filtration of the sharp Bragg reflections from the crystal can be easily removed during early data processing, ultimately enabling high-resolution reconstructions of the sample of interest11,12,13. Here, an optimized, step-by-step protocol is provided for robust production of streptavidin affinity grids and subsequent use in cryo-EM experiments. The protocol provided is expected to be completed over a 2-week period (Figure 1A). The first parts of the protocol describe the preparation of reagents, the pretreatment of grids, and the first carbon evaporation steps. Next, instructions are described for the preparation of the lipid monolayer and the growth of streptavidin crystals on EM grids. Additionally, instructions are provided for the use of streptavidin affinity grids in negative stain EM and cryo-EM experiments. Finally, procedures are provided for removing streptavidin-signal from micrographs once cryo-EM data has been acquired.
Our protocol describes how to make and use streptavidin affinity grids and how to process the data containing the streptavidin diffraction signal. There are several critical steps in the protocol that require special attention.
Unsuccessful grid batches can be traced back to several common errors. The most common source of error comes from using outdated or poor-quality reagents. It is especially important to prepare the biotinylated lipid solution exactly as described in the protocol. Further, any impurities, such as detergent residue on the labware or naturally present oils on skin, can affect the quality of the streptavidin crystals and, hence, the quality of the resulting images. It is therefore recommended to perform three washing cycles for the grids prior to the first carbon evaporation to remove possible surfactant contamination from the grid manufacturer (step 2.1). Additionally, contamination or impurity of the castor oil has been observed to hinder crystal formation on the grid.
Making and picking up the lipid monolayer is a task that should be practiced a few times to get a feeling for the small lipid volumes. It is not unusual for this step to fail the first two or three times before an adequate lipid monolayer can be produced, as is ultimately reflected in the quality of streptavidin crystals that are seen in negatively stained grids (Figure 4C).
It is important to never let the backside (gold side, lipid side) of the grid get wet during the grid fabrication process (steps 3.12-3.20). In the case the backside does get wet, it is recommended to discard the grid. This can result in the appearance of large lipid vesicles that disrupt the image quality (Figure 5D) (Row 3, Table 1). Lipid vesicles can also be observed due to insufficient washing after the lipid monolayer is applied (step 3.13). Three subsequent washes using crystallization buffer are recommended after touching the lipid monolayer prior to adding streptavidin.
After a thin layer of carbon has been evaporated onto trehalose-embedded grids, the backside of the grid can be wet without damaging the lattice quality. For example, wetting of the backside is frequently observed when the sample buffer contains even minimal amounts of detergent. Wetting of the backside by the sample can result in nonspecific binding of samples to the thin carbon film on the backside, which decreases the effectiveness of preventing particles from diffusing to the air-water interface or the use of affinity binding for on-grid purification strategies. If possible, add the sample to the grid in the absence of detergent. Detergent and other additives can be subsequently added during the grid washing steps or added in a final step before vitrification. This is one limitation to the use of streptavidin affinity grids; however, if the goal is to improve preferential orientation, sample preparation with buffers, including detergent, has been performed successfully11.
A common source of error can be traced to the age of the grids relative to both carbon evaporation steps (step 2.3 and step 3.21). In this protocol, the recommended waiting period is 5-7 days after the backside carbon evaporation before using streptavidin grids for cryo-EM. When grids are used too soon after fabrication, the lattice and lipid monolayer have been observed to mobilize out of the grid holes. A similar observation can be made when the grids are too old and used after six months (Figure 5A) (Row 1, Table 1). We hypothesize that this observation is explained by changes in the hydrophobicity of the carbon backing that is applied to stabilize the lipid monolayer and streptavidin crystals. Additionally, streptavidin lattices may appear mosaic (Figure 5B) (Row 3, Table 1) and broken in both negative-stain and cryo-EM if the monolayer is applied to grids where the first carbon evaporation layer (step 2.3)has not sufficiently aged.
An additional common source of error can be traced to the fragility of the streptavidin crystalline monolayer (the lipid monolayer is itself fluid and can reversibly expand or compress). The backside carbon evaporation (step 3.21) provides critical stability to both the monolayer and streptavidin lattice during the sample adsorption, washing, and cryo-EM blotting process. In the absence of sufficient carbon evaporation to the backside of the grid, streptavidin lattices will often appear fragmented after blotting/freezing (Figure 5C) (Row 2, Table 1). In this protocol, we suggest the use of a one-sided automated plunge freezer for single-sided blotting. This method yields highly reproducible blotting conditions that preserve the streptavidin lattice during the freezing process and allow for streamlined optimization of the sample application and blotting parameters. Alternatively, other automated plunge-freezing apparatuses have been used in combination with manual blotting. To do this, the actual blotting function is disabled in the device settings, and the grid is blotted instead by reaching with a pair of tweezers holding blotting paper through the side entry. This method can achieve high-quality results for laboratories without a single-sided blotting and plunge freezing device; however, grid-to-grid reproducibility is challenging.
While using streptavidin affinity grids has many advantages, some limitations of this method need to be taken into consideration. Owing to the nature of the procedure, it is usually not possible to assess the quality of the streptavidin lattice until the entire process has been completed. It is suggested to quickly screen the quality of each batch by negative stain before freezing samples. Due to the signal contributed by the streptavidin lattice to the raw images, it can be challenging in some cases to assess, from the images alone, whether there is a sufficient number of intact, disperse particles. For the same reason, on-the-fly processing of cryo-EM data during data acquisition is not possible unless the streptavidin signal subtraction procedure has been included in the data pre-processing pipeline. Because the streptavidin signal is usually subtracted from the motion-corrected images and not the frames themselves, Bayesian polishing, as implemented in popular software suites, may fail when using the subtraction procedures as described. It is therefore recommended to perform motion correction in multiple patches to minimize particle movement from the onset of data processing16.
Despite these limitations, streptavidin affinity grids offer many advantages. Two major advantages that streptavidin affinity grids provide over standard open-hole cryo-EM grids are a means to protect samples from the air-water interface and concentrate samples of low abundance (10-100 nM) on the grid. Other support layers, such as carbon and graphene oxide, can also be used as strategies to overcome these sample preparation bottlenecks. In one example, streptavidin affinity grids were the only solution to obtaining an intact reconstruction of a protein-nucleic acid interaction that was incompatible with cross-linking approaches.12
Another major advantage that streptavidin affinity grids provide is a solution to samples that adsorb to other support layers, such as carbon or graphene oxide, with preferred orientations that hinder the ability to obtain a 3D reconstruction of the sample of interest. Random biotinylation of samples using commercially available kits allows the random attachment of the sample of interest to the streptavidin monolayer to obtain more potential views to overcome this challenge.
Additionally, streptavidin affinity grids offer advantages that are unique to affinity-based grids. The high affinity and specificity of the streptavidin-biotin interaction allow the coupling of streptavidin affinity grids with other workflows that involve biotin to purify complexes of interest. In one published example, the authors immobilized a complex on streptavidin affinity grids and incubated a binding partner of unknown stoichiometry in large excess. After washing off any unbound protein, the correctly assembled super-complex was obtained and could be immediately analyzed with cryo-EM11. One possible future application may be to combine streptavidin-binding protein tags, the Avi-tag system, or proximity-labeling approaches with streptavidin affinity grids to extract single proteins and/or protein complexes directly from recombinant or endogenous sources without standard elaborate purification schemes.
By providing this protocol, labs should be able to easily reproduce the fabrication of streptavidin affinity grids and establish them as a more commonly used tool for structural analysis of protein complexes by cryo-EM.
The authors have nothing to disclose.
T.C. was supported by the National Institute of General Medical Sciences molecular biophysics training grant GM-08295 and the National Science Foundation graduate research fellowship under grant number DGE 2146752. R.G. and B.H. were supported by the National Institute of Health grant R21-GM135666 awarded to R.G. and B.H. This work was partially funded through the National Institute of General Medical Sciences grant R35-GM127018 awarded to E.N. E.N. is a Howard Hughes Medical Institute Investigator.
1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(biotinyl) sodium salt | Avanti Polar Lipids | 870285P | Comes as a powder. Dissolve at concentration of 1–10 mg/mL in chloroform/methanol/water solvent, 65:35:8 v/v and store in single use aliquots at -80 °C |
200 proof pure ethanol | Fisher Scientific | 07-678-005 | |
5 μL Hamilton Syringe | Hamilton | 87930 | 5 µL Microliter Syringe Model 75 RN, Small Removable Needle, 26 G, 2 in, point style 2 |
Castor Oil | Sigma Adrich | 259853 | |
Cell culture dishes untreated (35 mm) | Bio Basic | SP22146 | |
Chloroform | Sigma Aldrich | 650471 | |
D-(+)-Trehalose dihydrate,from starch, ≥99% | Sigma Aldrich | T9449 | |
DUMONT Anti-Capillary Reverse (self-closing) Tweezers Biology Grade | Ted Pella | 510-5NM | |
HPLC Grade Methanol | Fisher Scientific | A452-4 | |
Leica EM ACE600 High Vacuum Sputter Coater | Leica | ||
Leica EM GP2 Automatic Plunge Freezer | Leica | ||
Quantifoil R 2/1 Au300 | Quantifoil | Q84994 | |
Steptavidin | New England Bioscience | N7021S | Comes as 1 mg/mL solution. Dilute to final concentration of 0.5 mg /mL in crystallization bufferwith a final trehalose concentration of 10%. Can be flash frozen in 25–50 μL aliquots |
Talcum powder | Carolina | 896060 | |
Whatman Grade 1 filter paper | Cytiva | 1001-085 |