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Samples prepared for single-particle electron cryo-microscopy (cryo-EM) necessarily have a very high surface-to-volume ratio during the short period of time between thinning and vitrification. During this time, there is an obvious risk that macromolecules of interest may adsorb to the air–water interface with a preferred orientation, or that they may even become partially or fully unfolded at the interface. In addition, adsorption of macromolecules to an air–water interface may occur even before thinning. This paper addresses the question whether currently used methods of sample preparation might be improved if one could avoid such interfacial interactions. One possible way to do so might be to preemptively form a surfactant monolayer over the air–water interfaces, to serve as a structure-friendly slide and coverslip. An alternative is to immobilize particles of interest by binding them to some type of support film, which—to continue using the analogy—thus serves as a slide. In this case, the goal is not only to prevent the particles of interest from diffusing into contact with the air–water interface but also to increase the number of particles seen in each image. In this direction, it is natural to think of developing various types of affinity grids as structure-friendly alternatives to thin carbon films. Perhaps ironically, if precautions are not taken against adsorption of particles to air–water interfaces, sacrificial monolayers of denatured protein may take the roles of slide, coverslip, or even both.
Benjamin CJ, Wright KJ, Hyun S-H, Krynski K, Yu G, Bajaj R, Guo F, Stauffacher CV, Jiang W, Thompson DH, (2016) Nonfouling NTA-PEG-based TEM grid coatings for selective capture of histidine-tagged protein targets from cell lysates.Langmuir 32:551-559
Chamberlain AK, Handel TM, Marqusee S, (1996) Detection of rare partially folded molecules in equilibrium with the native conformation of RNaseH.Nat Struct Biol 3:782-787
Crucifix C, Uhring M, Schultz P, (2004) Immobilization of biotinylated DNA on 2-D streptavidin crystals.J Struct Biol 146:441-451
Frederik PM, Stuart MCA, Bomans PHH, Busing WM, (1989) Phospholipid, natures’s own slide and coverslip for cryo-electron microscopy.J Microsc-Oxf 153:81-92
Glaeser Robert M, Han B-G, Csencsits R, Killilea A, Pulk A, Cate Jamie HD, (2016) Factors that influence the formation and stability of thin, cryo-EM specimens.Biophys J 110:749-755
Han BG, Walton RW, Song A, Hwu P, Stubbs MT, Yannone SM, Arbelaez P, Dong M, Glaeser RM, (2012) Electron microscopy of biotinylated protein complexes bound to streptavidin monolayer crystals.J Struct Biol 180:249-253
Kelly DF, Abeyrathne PD, Dukovski D, Walz T, (2008) The affinity grid: a pre-fabricated EM grid for monolayer purification.J Mol Biol 382:423-433
Kelly DF, Dukovski D, Walz T, (2010) Strategy for the use of affinity grids to prepare non-his-tagged macromolecular complexes for single-particle electron microscopy.J Mol Biol 400:675-681
Llaguno MC, Xu H, Shi L, Huang N, Zhang H, Liu QH, Jiang QX, (2014) Chemically functionalized carbon films for single molecule imaging.J Struct Biol 185:405-417
Maity H, Maity M, Krishna MMG, Mayne L, Englander SW, (2005) Protein folding: the stepwise assembly of foldon units.Proc Natl Acad Sci USA 102:4741-4746
Quinn PJ, Dawson RMC, (1970) An analysis of interaction of protein with lipid monolayers at the air-water interface.Biochem J 116:671-680
Raffaini G, Ganazzoli F, (2010) Protein adsorption on a hydrophobic surface: a molecular dynamics study of lysozyme on graphite.Langmuir 26:5679-5689
Sosnick TR, Barrick D, (2011) The folding of single domain proteins—have we reached a consensus?.Curr Opin Struct Biol 21:12-24
Taylor KA, Glaeser RM, (2008) Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future.J Struct Biol 163:214-223
Trurnit HJ, (1960) A theory and method for the spreading of protein monolayers.J Colloid Sci 15:1-13
Wang LG, Ounjai P, Sigworth FJ, (2008) Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection.J Struct Biol 164:190-198
Yoshimura H, Scheybani T, Baumeister W, Nagayama K, (1994) 2-Dimensional protein array growth in thin layers of protein solution on aqueous subphases.Langmuir 10:3290-3295
Yu GM, Vago F, Zhang DS, Snyder JE, Yan R, Zhang C, Benjamin C, Jiang X, Kuhn RJ, Serwer P, Thompson DH, Jiang W, (2014) Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.J Struct Biol 187:1-9
Yu G, Li K, Jiang W, (2016) Antibody-based affinity cryo-EM grid.Methods 100:16-24
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