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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. Qie.Kuang@ki.se; Philip.Koeck@ki.se
*Corresponding authors. Qie.Kuang@ki.se; Philip.Koeck@ki.se
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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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