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A Refined Single-Particle Reconstruction Procedure to Process Two-Dimensional Crystal Images from Transmission Electron Microscopy

Published online by Cambridge University Press:  20 May 2015

Qie Kuang ,
Pasi Purhonen ,
Thirupathi Pattipaka ,
Yohannes H. Ayele ,
Hans Hebert  and
Philip J.B. Koeck
Qie Kuang*
Affiliation:
Karolinska Institutet, Department of Biosciences and Nutrition and KTH Royal Institute of Technology, School of Technology and Health, Novum, S-14183 Huddinge, Sweden
Pasi Purhonen
Affiliation:
Karolinska Institutet, Department of Biosciences and Nutrition and KTH Royal Institute of Technology, School of Technology and Health, Novum, S-14183 Huddinge, Sweden
Thirupathi Pattipaka
Affiliation:
Karolinska Institutet, Department of Biosciences and Nutrition and KTH Royal Institute of Technology, School of Technology and Health, Novum, S-14183 Huddinge, Sweden
Yohannes H. Ayele
Affiliation:
Karolinska Institutet, Department of Biosciences and Nutrition and KTH Royal Institute of Technology, School of Technology and Health, Novum, S-14183 Huddinge, Sweden
Hans Hebert
Affiliation:
Karolinska Institutet, Department of Biosciences and Nutrition and KTH Royal Institute of Technology, School of Technology and Health, Novum, S-14183 Huddinge, Sweden
Philip J.B. Koeck*
Affiliation:
Karolinska Institutet, Department of Biosciences and Nutrition and KTH Royal Institute of Technology, School of Technology and Health, Novum, S-14183 Huddinge, Sweden
*
*Corresponding authors. [email protected]; [email protected]
*Corresponding authors. [email protected]; [email protected]
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Abstract

Single-particle reconstruction (SPR) and electron crystallography (EC), two major applications in electron microscopy, can be used to determine the structure of membrane proteins. The three-dimensional (3D) map is obtained from separated particles in conventional SPR, but from periodic unit cells in EC. Here, we report a refined SPR procedure for processing 2D crystal images. The method is applied to 2D crystals of melibiose permease, a secondary transporter in Escherichia coli. The current procedure is improved from our previously published one in several aspects. The “gold standard Fourier shell correlation” resolution of our final reconstruction reaches 13 Å, which is significantly better than the previously obtained 17 Å resolution. The choices of different refinement parameters for reconstruction are discussed. Our refined SPR procedure could be applied to determine the structure of other membrane proteins in small or locally distorted 2D crystals, which are not ideal for EC.

Keywords

cryo-EMsingle-particle reconstructionelectron crystallographymembrane proteinsmelibiose permease
Type
Biological Applications and Techniques
Information
Microscopy and Microanalysis , Volume 21 , Issue 4 , August 2015 , pp. 876 - 885
Copyright
© Microscopy Society of America 2015 

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References

Crowther, R.A., Henderson, R. & Smith, J.M. (1996). MRC image processing programs. J Struct Biol 116, 9–16.CrossRefGoogle ScholarPubMed
Ethayathulla, A.S., Yousef, M.S., Amin, A., Leblanc, G., Kaback, H.R. & Guan, L. (2014). Structure-based mechanism for Na(+)/melibiose symport by MelB. Nat Commun 5, 3009.CrossRefGoogle ScholarPubMed
Fernandez, J.J., Luque, D., Caston, J.R. & Carrascosa, J.L. (2008). Sharpening high resolution information in single particle electron cryomicroscopy. J Struct Biol 164, 170–175.Google Scholar
Frank, J. (2006). Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in their Native State, pp. 34–62. Oxford and New York, NY: Oxford University Press.Google Scholar
Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M. & Leith, A. (1996). SPIDER and WEB: Processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116, 190–199.Google Scholar
Gipson, B., Zeng, X., Zhang, Z.Y. & Stahlberg, H. (2007). 2dx—User-friendly image processing for 2D crystals. J Struct Biol 157, 64–72.Google Scholar
Grigorieff, N. (2000). Resolution measurement in structures derived from single particles. Acta Crystallogr D Biol Crystallogr 56(Pt 10), 1270–1277.Google Scholar
Guan, L., Nurva, S. & Ankeshwarapu, S.P. (2011). Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium . J Biol Chem 286, 6367–6374.Google Scholar
Hacksell, I., Rigaud, J.L., Purhonen, P., Pourcher, T., Hebert, H. & Leblanc, G. (2002). Projection structure at 8 A resolution of the melibiose permease, an Na-sugar co-transporter from Escherichia coli . EMBO J 21, 3569–3574.Google Scholar
Henderson, R., Baldwin, J.M., Downing, K.H., Lepault, J. & Zemlin, F. (1986). Structure of purple membrane from Halobacterium halobium: Recording, measurement and evaluation of electron micrographs at 3.5 Å resolution. Ultramicroscopy 19, 147–178.Google Scholar
Koeck, P.J.B., Purhonen, P., Alvang, R., Grundberg, B. & Hebert, H. (2007). Single particle refinement in electron crystallography: A pilot study. J Struct Biol 160, 344–352.CrossRefGoogle ScholarPubMed
Kuang, Q., Purhonen, P., Jegerschold, C. & Hebert, H. (2014). The projection structure of Kch, a putative potassium channel in Escherichia coli, by electron crystallography. Biochim Biophys Acta 1838(1, Pt B), 237–243.Google Scholar
Kuang, Q., Purhonen, P., Jegerschold, C., Koeck, P.J. & Hebert, H. (2015). Free RCK arrangement in Kch, a putative Escherichia coli potassium channel, as suggested by electron crystallography. Structure 23, 199–205.Google Scholar
Ludtke, S.J., Baldwin, P.R. & Chiu, W. (1999). EMAN: Semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128, 82–97.CrossRefGoogle ScholarPubMed
Milkman, R. (1994). An Escherichia coli homologue of eukaryotic potassium channel proteins. Proc Natl Acad Sci USA 91, 3510–3514.Google Scholar
Mindell, J.A. & Grigorieff, N. (2003). Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol 142, 334–347.Google Scholar
Mizushima, K., Awakihara, S., Kuroda, M., Ishikawa, T., Tsuda, M. & Tsuchiya, T. (1992). Cloning and sequencing of the melB gene encoding the melibiose permease of Salmonella typhimurium LT2. Mol Gen Genet 234, 74–80.CrossRefGoogle ScholarPubMed
Penczek, P.A. (2010). Resolution measures in molecular electron microscopy. Methods Enzymol 482, 73–100.Google Scholar
Perkins, G.A., Downing, K.H. & Glaeser, R.M. (1995). Crystallographic extraction and averaging of data from small image areas. Ultramicroscopy 60, 283–294.Google Scholar
Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C. & Ferrin, T.E. (2004). UCSF Chimera—A visualization system for exploratory research and analysis. J Comput Chem 25, 1605–1612.Google Scholar
Purhonen, P., Lundback, A.K., Lemonnier, R., Leblanc, G. & Hebert, H. (2005). Three-dimensional structure of the sugar symporter melibiose permease from cryo-electron microscopy. J Struct Biol 152, 76–83.Google Scholar
Rosenthal, P.B. & Henderson, R. (2003). Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol 333, 721–745.Google Scholar
Scherer, S., Arheit, M., Kowal, J., Zeng, X. & Stahlberg, H. (2014). Single particle 3D reconstruction for 2D crystal images of membrane proteins. J Struct Biol 185, 267–277.CrossRefGoogle ScholarPubMed
Scheres, S.H., Gao, H., Valle, M., Herman, G.T., Eggermont, P.P., Frank, J. & Carazo, J.M. (2007). Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nat Methods 4, 27–29.Google Scholar
Sorzano, C.O., Jonic, S., El-Bez, C., Carazo, J.M., De Carlo, S., Thevenaz, P. & Unser, M. (2004). A multiresolution approach to orientation assignment in 3D electron microscopy of single particles. J Struct Biol 146, 381–392.Google Scholar
Stoylova, S.S., Ford, R.C. & Holzenburg, A. (1999). Cryo-electron crystallography of small and mosaic 2-D crystals: An assessment of a procedure for high-resolution data retrieval. Ultramicroscopy 77, 113–128.Google Scholar
Tang, G., Peng, L., Baldwin, P.R., Mann, D.S., Jiang, W., Rees, I. & Ludtke, S.J. (2007). EMAN2: An extensible image processing suite for electron microscopy. J Struct Biol 157, 38–46.Google Scholar
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