title: 2D and 3D crystallization of the wild-type IIC domain of the glucose PTS transporter from Escherichia coli.
abstract: The bacterial phosphoenolpyruvate: sugar phosphotransferase system serves the combined uptake and phosphorylation of carbohydrates. This structurally and functionally complex system is composed of several conserved functional units that, through a cascade of phosphorylated intermediates, catalyze the transfer of the phosphate moiety from phosphoenolpyruvate to the substrate, which is bound to the integral membrane domain IIC. The wild-type glucose-specific IIC domain (wt-IIC(glc)) of Escherichia coli was cloned, overexpressed and purified for biochemical and functional characterization. Size-exclusion chromatography and scintillation-proximity binding assays showed that purified wt-IIC(glc) was homogenous and able to bind glucose. Crystallization was pursued following two different approaches: (i) reconstitution of wt-IIC(glc) into a lipid bilayer by detergent removal through dialysis, which yielded tubular 2D crystals, and (ii) vapor-diffusion crystallization of detergent-solubilized wt-IIC(glc), which yielded rhombohedral 3D crystals. Analysis of the 2D crystals by cryo-electron microscopy and the 3D crystals by X-ray diffraction indicated resolutions of better than 6Å and 4Å, respectively. Furthermore, a complete X-ray diffraction data set could be collected and processed to 3.93Å resolution. These 2D and 3D crystals of wt-IIC(glc) lay the foundation for the determination of the first structure of a bacterial glucose-specific IIC domain.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Kalbermatter
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Jeckelmann
authors.affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Po-Lin
authors.initials: PL
authors.lastname: Chiu
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
authors.forename: Zöhre
authors.initials: Z
authors.lastname: Ucurum
authors.affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; Howard Hughes Medical Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Walz
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland. Electronic address: dimitrios.fotiadis@ibmm.unibe.ch.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fotiadis
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001692:Biological Transport / Q000502:physiology; D003460:Crystallization / Q000379:methods; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D051246:Glucose Transport Proteins, Facilitative / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D051246:Glucose Transport Proteins, Facilitative; D008565:Membrane Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D005947:Glucose
keywords: Cryo-electron microscopy; Membrane protein; Scintillation-proximity assay; Three-dimensional crystal; Transport protein; Two-dimensional crystal; X-ray crystallography