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.

Fabrication of Streptavidin Affinity Grids for Cryo-EM Structural Studies

<ol>
	<li>Glaeser, R. M., Nogales, E., Chiu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-particle Cryo-EM of Biological Macromolecules. IOP Publishing. , (2021).</li><li>Carragher, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+outcomes+when+optimizing+'standard'+sample+preparation+for+single-particle+cryo-EM.">Current outcomes when optimizing 'standard' sample preparation for single-particle cryo-EM.</a> Journal of Microscopy. 276 (1), 39-45 (2019).</li><li>Drulyte, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+altering+particle+distributions+in+cryo-electron+microscopy+sample+preparation.">Approaches to altering particle distributions in cryo-electron microscopy sample preparation.</a> Acta Crystallographica Section D-Structural Biology. 74, 560-571 (2018).</li><li>Noble, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+single+particle+cryoEM+sample+and+grid+characterization+by+tomography.">Routine single particle cryoEM sample and grid characterization by tomography.</a> eLife. 7, e34257 (2018).</li><li>Patel, A., Toso, D., Litvak, A., Nogales, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+graphene+oxide+coating+improves+cryo-EM+sample+preparation+and+data+collection+from+tilted+grids.">Efficient graphene oxide coating improves cryo-EM sample preparation and data collection from tilted grids.</a> bioRxiv. , (2021).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+and+robust+covalently+linked+graphene+oxide+affinity+grids+for+high-resolution+cryo-EM.">General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM.</a> Proceedings of the National Academy of Sciences. 117 (39), 24269-24273 (2020).</li><li>Pantelic, R. S., Meyer, J. C., Kaiser, U., Baumeister, W., Plitzko, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graphene+oxide:+A+substrate+for+optimizing+preparations+of+frozen-hydrated+samples.">Graphene oxide: A substrate for optimizing preparations of frozen-hydrated samples.</a> Journal of Structural Biology. 170 (1), 152-156 (2010).</li><li>Williams, R. C., Glaeser, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+carbon+support+films+for+electron+microscopy.">Ultrathin carbon support films for electron microscopy.</a> Science. 175 (4025), 1000-1001 (1972).</li><li>Han, B. -. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+shelf-life+streptavidin+support-films+suitable+for+electron+microscopy+of+biological+macromolecules.">Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules.</a> Journal of Structural Biology. 195 (2), 238-244 (2016).</li><li>Han, B. -. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+biotinylated+protein+complexes+bound+to+streptavidin+monolayer+crystals.">Electron microscopy of biotinylated protein complexes bound to streptavidin monolayer crystals.</a> Journal of Structural Biology. 180 (1), 249-253 (2012).</li><li>Dom&#237;nguez-Mart&#237;n, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+a+phycobilisome+in+light-harvesting+and+photoprotected+states.">Structures of a phycobilisome in light-harvesting and photoprotected states.</a> Nature. 609 (7928), 835-845 (2022).</li><li>Kasinath, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JARID2+and+AEBP2+regulate+PRC2+in+the+presence+of+H2AK119ub1+and+other+histone+modifications.">JARID2 and AEBP2 regulate PRC2 in the presence of H2AK119ub1 and other histone modifications.</a> Science. 371 (6527), eabc3393 (2021).</li><li>Lahiri, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3.1+structure+of+yeast+RNA+polymerase+II+elongation+complex+stalled+at+a+cyclobutane+pyrimidine+dimer+lesion+solved+using+streptavidin+affinity+grids.">3.1 structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids.</a> Journal of Structural Biology. 207 (3), 270-278 (2019).</li><li>Marr, C. R., Benlekbir, S., Rubinstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+carbon+films+with+~+500nm+holes+for+cryo-EM+with+a+direct+detector+device.">Fabrication of carbon films with ~ 500nm holes for cryo-EM with a direct detector device.</a> Journal of Structural Biology. 185 (1), 42-47 (2014).</li><li>Levine, M. J., Schwarz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+guidelines+for+producing+molecular+assemblies+by+Langmuir-Blodgett+techniques.">Experimental guidelines for producing molecular assemblies by Langmuir-Blodgett techniques.</a> Journal of Chemical Education. 65 (7), 638 (1988).</li><li>Zheng, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MotionCor2:+anisotropic+correction+of+beam-induced+motion+for+improved+cryo-electron+microscopy.">MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy.</a> Nature Methods. 14 (4), 331-332 (2017).</li></ol>

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&#160;(Figure 5A)&#160;(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)&#160;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)&#160;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)&#160;(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.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(biotinyl) sodium salt&#160;</td>
			<td>Avanti Polar Lipids</td>
			<td>870285P</td>
			<td>Comes as a powder. Dissolve at concentration of 1&#8211;10 mg/mL&#160; in chloroform/methanol/water solvent, 65:35:8 v/v and store in single use aliquots at -80 &#176;C</td>
		</tr>
		<tr>
			<td>200 proof pure ethanol</td>
			<td>Fisher Scientific</td>
			<td>07-678-005</td>
			<td></td>
		</tr>
		<tr>
			<td>5 &#956;L Hamilton Syringe&#160;</td>
			<td>Hamilton</td>
			<td>87930</td>
			<td>5 &#181;L Microliter Syringe Model 75 RN, Small Removable Needle, 26 G, 2 in, point style 2</td>
		</tr>
		<tr>
			<td>Castor Oil</td>
			<td>Sigma Adrich</td>
			<td>259853</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dishes untreated (35 mm)</td>
			<td>Bio Basic</td>
			<td>SP22146</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform&#160;</td>
			<td>Sigma Aldrich</td>
			<td>650471</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Trehalose dihydrate,from starch, &#8805;99%</td>
			<td>Sigma Aldrich</td>
			<td>T9449</td>
			<td></td>
		</tr>
		<tr>
			<td>DUMONT Anti-Capillary Reverse (self-closing) Tweezers Biology Grade</td>
			<td>Ted Pella</td>
			<td>510-5NM</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Grade Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica EM ACE600 High Vacuum Sputter Coater</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica EM GP2 Automatic Plunge Freezer</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quantifoil R 2/1 Au300&#160;</td>
			<td>Quantifoil</td>
			<td>Q84994</td>
			<td></td>
		</tr>
		<tr>
			<td>Steptavidin</td>
			<td>New England Bioscience</td>
			<td>N7021S</td>
			<td>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&#8211;50 &#956;L aliquots</td>
		</tr>
		<tr>
			<td>Talcum powder&#160;</td>
			<td>Carolina</td>
			<td>896060</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Grade 1 filter paper</td>
			<td>Cytiva</td>
			<td>1001-085</td>
			<td></td>
		</tr>
	</tbody>
</table>

streptavidin-affinity grid fabrication for cryo-electron microscopy sample preparation

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.

After the carbon evaporation in step 3.21 (usually after one week), streptavidin affinity grids can be used for cryo-EM or negative-stain sample preparation following the procedures described in steps 4 and 5. A representative micrograph captured using a K3 detector on a titan Krios at 81,000X magnification with a pixel size of 1.05 &#197; of a biotinylated sample frozen with streptavidin affinity grids is shown in Figure 4A. Successful lattice formation is observed by the continuous grid pattern that appears in the background of the image. This may be more easily observed by the diffraction pattern that appears in the fast Fourier transform (FFT) shown in Figure 4A (right). After data collection, following the procedure outlined in step 6 of this protocol, the signal that is contributed to the image by the streptavidin lattice can be computationally masked to produce a subtracted micrograph (Figure 4B) that can be used for subsequent data processing steps. The FFT in Figure 4B (right)&#160;shows that the diffraction pattern observed in Figure 4A (right) has been successfully removed from the original image.
Figure 4C shows an example micrograph taken on a Tecnai 12 microscope at a magnification of 49,000x with a pixel size of 1.6 &#197; of a biotinylated sample bound to streptavidin affinity grids and negatively stained using uranyl formate. Grids were prepared following the procedure outlined in step 4 of this protocol, leaving a thicker film of stain.
There are several common observations when the streptavidin grid fabrication procedure is unsuccessful that are described further in the discussion section and Table 1. Figure 5 shows several examples of these observations. Figure 5A shows a micrograph of a negatively stained streptavidin affinity grid used from a batch made over six months old. The monolayer has mobilized from the holes in the carbon foil of the quantifoil grids and is observed with similar dimensions to the holes in the grid. Figure 5B shows an example of streptavidin crystals that have high mosaicity that become easily perturbed during sample preparation procedures. Figure 5C shows a representative micrograph from a streptavidin affinity grid in which the carbon evaporation in step 3.21 is too thin or omitted. The streptavidin lattice has become fragmented during the cryo-EM sample preparation process. Figure 5D shows a negatively stained streptavidin affinity grid that has contamination from lipid vesicles, likely due to insufficient washes during step 3.13 or the gold side of the grid becoming wet during steps 3.13-3.20. Please refer to the discussion section and Table 1 for strategies to overcome these common issues.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66197/66197fig01.jpg" /> 
Figure 1: Schematic of streptavidin affinity grid fabrication procedure. (A) Overview timeline of the streptavidin affinity grid fabrication procedure. The entire procedure can be completed over two weeks requiring two carbon evaporation steps and one week after each to allow the carbon to sufficiently age. (B)&#160;Zoomed in view of a grid square of a streptavidin affinity grid showing the layers that make up the grid after completion of the fabrication process, including the standard cryo-EM grid with gold bars and holey-carbon film, the first evaporated carbon layer, lipid monolayer, 2D-streptavidin crystal, and the backside evaporated carbon support layer. <a href="https://www.jove.com/files/ftp_upload/66197/66197fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66197/66197fig02.jpg" /> 
Figure 2: Lipid monolayer and streptavidin crystallization (A) Images demonstrating how to successfully form the lipid monolayer within the small Petri dish utilizing talcum powder to create a boundary for the castor oil that creates constant surface pressure while forming the lipid monolayer. (B) Images demonstrating how to grow streptavidin crystals onto cryo-EM grids. First, the carbon side of the grids is touched to the lipid monolayer and followed by three consecutive washes in the crystallization buffer. Streptavidin is added, and grids are incubated in a humidity chamber for 2 h. <a href="https://www.jove.com/files/ftp_upload/66197/66197fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66197/66197fig03.jpg" /> 
Figure 3: Example parameters for the process_subtraction.cfg file for performing streptavidin lattice subtraction from micrographs (step 6). <a href="https://www.jove.com/files/ftp_upload/66197/66197fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66197/66197fig04.jpg" /> 
Figure 4: Representative results of successful streptavidin affinity grid fabrication. (A)&#160;Cryo-EM micrograph of biotinylated protein/nucleosome complexes prepared with homemade streptavidin affinity grids. The FFT is shown on the right and shows the streptavidin crystal diffraction pattern. (B)&#160;Same micrograph as in panel A is shown following the lattice subtraction procedure to remove the signal from the 2D streptavidin crystal from the original image. (C)&#160;An example of a micrograph obtained from negative-staining of a biotinylated sample bound to streptavidin-affinity grids. <a href="https://www.jove.com/files/ftp_upload/66197/66197fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66197/66197fig05.jpg" /> 
Figure 5: Representative results of unsuccessful streptavidin affinity grid fabrication. (A) Micrograph showing the mobilization of the streptavidin monolayer from the grid holes when grids are older than six months. (B)&#160;Micrograph showing streptavidin lattices with high mosaicity that are easily damaged during both cryo-EM and negative-stain sample preparation procedures. (C)&#160;Micrograph showing fragmentation of the streptavidin lattice during cryo-EM sample preparation when the carbon evaporation layer in step 3.21 is too thin or omitted. (D) Representative micrograph that is contaminated with lipid vesicles likely due to insufficient washing during step 3.13 or the gold side of the grid becoming wet during steps 3.13-3.20. <a href="https://www.jove.com/files/ftp_upload/66197/66197fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Troubleshooting guide to overcome commonly experienced challenges that are encountered during unsuccessful streptavidin affinity grid fabrication. <a href="https://www.jove.com/files/ftp_upload/66197/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental Coding File 1: lsub.m <a href="https://www.jove.com/files/ftp_upload/66197/lsub.zip" target="_blank">Please click here to download this File.</a>
Supplemental Coding File 2: process_subtration.sh <a href="https://www.jove.com/files/ftp_upload/66197/process_subtraction.zip" target="_blank">Please click here to download this File.</a>
Supplemental Coding File 3: process_subtraction.cfg <a href="https://www.jove.com/files/ftp_upload/66197/process_subtraction3.zip" target="_blank">Please click here to download this File.</a>
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Watch this Scientific Journal Video about Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation at JoVE.com

Author Spotlight: Enhancing Cryo-EM Sample Preparation with Streptavidin-Biotin Approach

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 Grid Fabrication for Cryo-Electron Microscopy Sample Preparation

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, ...

streptavidin-affinity-grid-fabrication-for-cryo-electron-microscopy

Research

JoVE Journal

Biochemistry

5.1K Views.  University of California, Berkeley. 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.

Video: Author Spotlight: Enhancing Cryo-EM Sample Preparation with Streptavidin-Biotin Approach

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown into highly ordered two-dimensional (2D) crystals under favorable conditions. The quality of the grown 2D crystals is crucial to the resolution of the final reconstruction via 2D image processing. Over the years, lipid monolayers have been used as a supporting layer to foster the 2D crystallization of peripheral membrane proteins as well as soluble proteins. This method can also be applied to 2D crystallization of integral membrane proteins but requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the monolayer. A lipid monolayer forms at the air-water interface such that the polar lipid head groups remain hydrated in the aqueous phase and the non-polar, acyl chains, tails partition into the air, breaking the surface tension and flattening the water surface. The charged nature or distinctive chemical moieties of the head groups provide affinity for proteins in solution, promoting binding for 2D array formation. A newly formed monolayer with the 2D array can be readily transfer into an electron microscope (EM) on a carbon-coated copper grid used to lift and support the crystalline array. In this work, we describe a lipid monolayer methodology for cryogenic electron microscopic (cryo-EM) imaging.

Lipid monolayers have been used as a foundation for forming two-dimensional (2D) protein crystals for structural studies for decades. They are stable at the air-water interface and can serve as a thin supporting material for electron imaging. Here we present the proven steps on preparing lipid monolayers for biological studies.

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown ...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method for structural biologists to determine high-resolution structures of a wide variety of macromolecules. Multiple software suites are available to new and expert users for image processing and structure calculation, which streamline the same basic workflow: movies acquired by the microscope detectors undergo correction for beam-induced motion and contrast transfer function (CTF) estimation. Next, particle images are selected and extracted from averaged movie frames for iterative 2D and 3D classification, followed by 3D reconstruction, refinement, and validation. Because various software packages employ different algorithms and require varying levels of expertise to operate, the 3D maps they generate often differ in quality and resolution. Thus, users regularly transfer data between a variety of programs for optimal results. This paper provides a guide for users to navigate a workflow across the popular software packages: cryoSPARC v3, RELION-3, and Scipion 3 to obtain a near-atomic resolution structure of the adeno-associated virus (AAV). We first detail an image processing pipeline with cryoSPARC v3, as its efficient algorithms and easy-to-use GUI allow users to quickly arrive at a 3D map. In the next step, we use PyEM and in-house scripts to convert and transfer particle coordinates from the best quality 3D reconstruction obtained in cryoSPARC v3 to RELION-3 and Scipion 3 and recalculate 3D maps. Finally, we outline steps for further refinement and validation of the resultant structures by integrating algorithms from RELION-3 and Scipion 3. In this article, we describe how to effectively utilize three processing platforms to create a single and robust workflow applicable to a variety of data sets for high-resolution structure determination.

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

This article describes how to effectively utilize three cryo-EM processing platforms, i.e., cryoSPARC v3, RELION-3, and Scipion 3, to create a single and robust workflow applicable to a variety of single-particle data sets for high-resolution structure determination.

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...

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

Summary

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Chapters in this video

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Preparation of High-Temperature Sample Grids for Cryo-EM

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion