Technical Note Electron microscopy of biotinylated protein complexes bound to streptavidin monolayer crystals Bong-Gyoon Han a , Ross W. Walton a , Amos Song a , Peter Hwu a , Milton T. Stubbs b , Steven M. Yannone a , Pablo Arbeláez c , Ming Dong d , Robert M. Glaeser a,⇑ a Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA, 94720, USA b Institute for Biotechnology, Martin-Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany c Department of Electrical Engineering and Computer Science, University of California, Berkeley, CA, 94720, USA d Genomics Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA, 94720, USA article info Article history: Received 15 January 2012 Received in revised form 7 April 2012 Accepted 23 April 2012 Available online xxxx Keywords: Cryo-EM Specimen preparation Biotinylation Streptavidin abstract Chemical biotinylation of protein complexes followed by binding to two-dimensional (monolayer) crys- tals of streptavidin is shown to be an effective way to prepare cryo-EM specimens from samples at low protein concentration. Three different multiprotein complexes are used to demonstrate the generality of this method. In addition, native thermosomes, purified from Sulfolobus solfataricus P2, are used to dem- onstrate that a uniform distribution of Euler angles is produced, even though this particle is known to adopt a preferred orientation when other methods of cryo-EM specimen preparation are used. Ó 2012 Elsevier Inc. All rights reserved. Preparation of specimen grids for cryo-EM generally involves blotting excess sample that has been applied either to a holey car- bon film or to a thin, continuous carbon film, followed by plunging the grid into liquid ethane (Dobro et al., 2010). Biological macro- molecules retained in the resulting thin, aqueous film are expected to collide with the air–water interface (or the thin carbon film, if any) a thousand times or more in the interval between blotting and freezing (Taylor and Glaeser, 2008). The preparation of cryo- EM samples might thus be improved by immobilizing particles on a supporting surface with properties similar to those of materi- als used for affinity chromatography. Affinity-based binding to derivatized lipids has been used for many years to produce both monolayer crystals and ordered helical arrays of biological macromolecules (Glaeser et al., 2007; Wilson-Kubalek et al., 2010). It has only recently been the case, however, that lipid monolayers have been used to prepare samples for single-particle cryo-EM (Kelly et al., 2008a,b, 2010a,b). In addi- tion, monolayer crystals of streptavidin have been used as affinity substrates for binding DNA, end-labeled with biotin, which was then used as bait for RNA polymerase (Crucifix et al., 2004). More recently, monolayer crystals of streptavidin have also been used to bind biotinylated proteoliposomes (Wang and Sigworth, 2009, 2010; Wang et al., 2008). In a related application, an attempt was made to use monolayer crystals of streptavidin as a template for two-dimensional crystallization of a second, biotinylated pro- tein, but only randomly distributed (single) particles were ob- tained (Darst et al., 1991). Following up on the earlier work of Darst et al. (1991), we reconsider the use of streptavidin-monolayer crystals to bind chemically biotinylated, soluble-protein complexes. In our case, however, the intended goal is to improve sample preparation for single-particle electron microscopy. We first show, using nega- tively stained samples of three different protein complexes, that this is a broadly general way to immobilize particles onto the EM grid. We also show that the particle density remains high even when a sample concentration as low as 10 lg/ml is used to pre- pare unstained, cryo-EM specimens. Finally, we use cryo-EM images of the thermosome from Sulfolobus solfataricus P2 to dem- onstrate that a uniform distribution of Euler angles is produced. 1. The degree of biotinylation can be characterized spectrophotometrically Three different protein complexes were used for the current work. Horse spleen apoferritin (MW 480 kDa) was purchased from Sigma. Thermosomes (MW1.17 MDa) were purified from Sulfolo- bus solfataricus P2. Briefly, biomass was homogenized by pressure 1047-8477/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsb.2012.04.025 ⇑ Corresponding author. Address: 363B Donner Laboratory, Lawrence Berkeley National Laboratory, University of California, Berkeley CA 94720, USA. Fax: +1 510 486 6488. E-mail address: rmglaeser@lbl.gov (R.M. Glaeser). Journal of Structural Biology xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi Please cite this article in press as: Han, B.-G., et al. Electron microscopy of biotinylated protein complexes bound to streptavidin monolayer crystals. J. Struct. Biol. (2012), http://dx.doi.org/10.1016/j.jsb.2012.04.025