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

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	<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. -. 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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. 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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><tbody><tr><td>1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(biotinyl) sodium salt&amp;nbsp;</td><td>Avanti Polar Lipids</td><td>870285P</td><td>Comes as a powder. Dissolve at concentration of 1&amp;ndash;10 mg/mL&amp;nbsp; in chloroform/methanol/water solvent, 65:35:8 v/v and store in single use aliquots at -80 &amp;deg;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 &amp;mu;L Hamilton Syringe&amp;nbsp;</td><td>Hamilton</td><td>87930</td><td>5 &amp;micro;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&amp;nbsp;</td><td>Sigma Aldrich</td><td>650471</td><td></td></tr><tr><td>D-(+)-Trehalose dihydrate,from starch, &amp;ge;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&amp;nbsp;</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&amp;ndash;50 &amp;mu;L aliquots</td></tr><tr><td>Talcum powder&amp;nbsp;</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>
Supplemental Coding File 4: bg_drill_hole.m <a href="https://www.jove.com/files/ftp_upload/66197/bg_drill_hole.zip" target="_blank">Please click here to download this File.</a>
Supplemental Coding File 5: bg_FastSubtract_standard.m <a href="https://www.jove.com/files/ftp_upload/66197/bg_FastSubtract_standard.zip" target="_blank">Please click here to download this File.</a>
Supplemental Coding File 6: bg_Pick_Amp_masked_standard.m <a href="https://www.jove.com/files/ftp_upload/66197/bg_Pick_Amp_masked_standard.zip" target="_blank">Please click here to download this File.</a>
Supplemental Coding File 7: bg_push_by_rot.m <a href="https://www.jove.com/files/ftp_upload/66197/bg_push_by_rot.zip" target="_blank">Please click here to download this File.</a>
Supplemental Coding File 8: ReadMRC.m <a href="https://www.jove.com/files/ftp_upload/66197/ReadMRC.zip" target="_blank">Please click here to download this File.</a>
Supplemental Coding File 9: WriteMRC.m <a href="https://www.jove.com/files/ftp_upload/66197/WriteMRC.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

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Immunology and Infection

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, ...

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers of multi-layered samples. This removal of layers prior to plunge freezing can aid in reducing sample thickness to a level suitable for cryo-EM data collection, improving sample flatness, and facilitating image processing. The peel-blot technique allows for the separation of multilamellar membranes into single layers, of layered 2D crystals into individual crystals, and of stacked, sheet-like structures of soluble proteins to likewise be separated into single layers. The high sample thickness of these types of samples frequently poses insurmountable problems for cryo-EM data collection and cryo-EM image processing, especially when the microscope stage must be tilted for data collection. Furthermore, grids of high concentrations of any of these samples can be prepared for efficient data collection since sample concentration prior to grid preparation can be increased and the peel-blot technique adjusted to result in a dense distribution of single-layered specimen.

The peel-blot technique is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...

Coating CryoEM Grids with Monolayer Graphene to Improve Cryo-Sample Preparation

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 &#181;m wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM&#39;s widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 &#197; resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

Author Spotlight: Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids 

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...

Preparation of Electron Microscopy Grids for In Situ Cellular Cryotomography

Sample Preparation for In Situ Cryotomography of Mammalian Cells

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly relevant to cellular biology and virology. The potential of combining cryotomography with other microscopy modalities makes it a perfect technique for integrative and correlative imaging. However, sample preparation for in situ cellular tomography is not straightforward, as cells do not readily attach and stretch over the electron microscopy grid. Additionally, the grids themselves are fragile and can break if handled too forcefully, resulting in the loss of imageable areas. The geometry of tissue culture dishes can also pose a challenge when manipulating the grids with tweezers. Here, we describe the tips and tricks to overcome these (and other) challenges and prepare good-quality samples for in situ cellular cryotomography and correlative imaging of adherent mammalian cells. With continued advances in cryomicroscopy technology, this technique holds enormous promise for advancing our understanding of complex biological systems.

Author Spotlight: Optimizing Grid Preparation for Enhanced Cryoelectron Tomography 

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native conditions, suspended in vitreous ice. This technique allows for the high-resolution visualization of proteins and other biomolecules without the need for crystallization, offering significant insights into their function and mechanism. Recent advancements in single-particle analysis, coupled with improved computational data processing, have made cryo-EM an indispensable tool in modern structural biology. Despite its growing adoption, cryo-EM faces persistent challenges that can limit its effectiveness, particularly uneven particle distribution. This issue often leads to poor resolution and reduced accuracy in reconstructed protein structures. This article outlines a simple, practical approach to address this challenge, using the small heat-shock protein from Methanocaldococcus jannaschii (MjsHSP16.5) as an example. The method optimizes sample preparation to minimize preferential adsorption, ensuring more homogeneous particle distribution and higher-quality protein cryo-EM structures. This technique offers valuable guidance for researchers aiming to overcome similar challenges in structural studies.

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biology

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, ...