title: A Refined Single-Particle Reconstruction Procedure to Process Two-Dimensional Crystal Images from Transmission Electron Microscopy.
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.
authors.affiliation: Karolinska Institutet,Department of Biosciences and Nutrition and KTH Royal Institute of Technology,School of Technology and Health,Novum,S-14183 Huddinge,Sweden.
authors.forename: Qie
authors.initials: Q
authors.lastname: Kuang
authors.affiliation: Karolinska Institutet,Department of Biosciences and Nutrition and KTH Royal Institute of Technology,School of Technology and Health,Novum,S-14183 Huddinge,Sweden.
authors.forename: Pasi
authors.initials: P
authors.lastname: Purhonen
authors.affiliation: Karolinska Institutet,Department of Biosciences and Nutrition and KTH Royal Institute of Technology,School of Technology and Health,Novum,S-14183 Huddinge,Sweden.
authors.forename: Thirupathi
authors.initials: T
authors.lastname: Pattipaka
authors.affiliation: Karolinska Institutet,Department of Biosciences and Nutrition and KTH Royal Institute of Technology,School of Technology and Health,Novum,S-14183 Huddinge,Sweden.
authors.forename: Yohannes H
authors.initials: YH
authors.lastname: Ayele
authors.affiliation: Karolinska Institutet,Department of Biosciences and Nutrition and KTH Royal Institute of Technology,School of Technology and Health,Novum,S-14183 Huddinge,Sweden.
authors.forename: Hans
authors.initials: H
authors.lastname: Hebert
authors.affiliation: Karolinska Institutet,Department of Biosciences and Nutrition and KTH Royal Institute of Technology,School of Technology and Health,Novum,S-14183 Huddinge,Sweden.
authors.forename: Philip J B
authors.initials: PJ
authors.lastname: Koeck
mesh_terms: D003461:Crystallography / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D027981:Symporters / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D027981:Symporters; C021548:melibiose permease
keywords: cryo-EM; electron crystallography; melibiose permease; membrane proteins; single-particle reconstruction