TY - JOUR
T1 - 2D and 3D crystallization of the wild-type IIC domain of the glucose PTS transporter from Escherichia coli
AU - Kalbermatter, David
AU - Jeckelmann, Jean Marc
AU - Chiu, Po Lin
AU - Ucurum, Zöhre
AU - Walz, Thomas
AU - Fotiadis, Dimitrios
N1 - Funding Information:
We thank Dr. D. Harder for help with the SPA experiments. X-ray diffraction data were collected at the Swiss Light Source (SLS) of the Paul Scherrer Institute (Villigen, Switzerland), and we thank the staff of the X06SA beamline, especially Drs. T. Tomizaki and M. Wang, for excellent technical support. We gratefully acknowledge financial support to D.F. from the University of Bern , the Swiss National Science Foundation (Grant 31003A_144168 ), the Bern University Research Foundation , and the National Centre of Competence in Research (NCCR) TransCure and NCCR Molecular Systems Engineering . T.W. is an investigator with the Howard Hughes Medical Institute.
PY - 2015/9/1
Y1 - 2015/9/1
N2 - 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.
AB - 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.
KW - Cryo-electron microscopy
KW - Membrane protein
KW - Scintillation-proximity assay
KW - Three-dimensional crystal
KW - Transport protein
KW - Two-dimensional crystal
KW - X-ray crystallography
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U2 - 10.1016/j.jsb.2015.08.003
DO - 10.1016/j.jsb.2015.08.003
M3 - Article
C2 - 26260226
AN - SCOPUS:84940614215
SN - 1047-8477
VL - 191
SP - 376
EP - 380
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
M1 - 6753
ER -