2D and 3D crystallization of the wild-type IIC domain of the glucose PTS transporter from Escherichia coli

David Kalbermatter, Jean Marc Jeckelmann, Po-Lin Chiu, Zöhre Ucurum, Thomas Walz, Dimitrios Fotiadis

Research output: Contribution to journalArticle

3 Scopus citations

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-IICglc) 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-IICglc was homogenous and able to bind glucose. Crystallization was pursued following two different approaches: (i) reconstitution of wt-IICglc into a lipid bilayer by detergent removal through dialysis, which yielded tubular 2D crystals, and (ii) vapor-diffusion crystallization of detergent-solubilized wt-IICglc, 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-IICglc lay the foundation for the determination of the first structure of a bacterial glucose-specific IIC domain.

Original languageEnglish (US)
Article number6753
Pages (from-to)376-380
Number of pages5
JournalJournal of Structural Biology
Volume191
Issue number3
DOIs
StatePublished - Sep 1 2015
Externally publishedYes

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Keywords

  • Cryo-electron microscopy
  • Membrane protein
  • Scintillation-proximity assay
  • Three-dimensional crystal
  • Transport protein
  • Two-dimensional crystal
  • X-ray crystallography

ASJC Scopus subject areas

  • Structural Biology
  • Medicine(all)

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