Computational protocol: A bacterial ABC transporter enables import of mammalian host glycosaminoglycans

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Protocol publication

[…] To determine the three-dimensional structure of Smon0123, the purified protein was crystallized by sitting drop vapor diffusion. The 1 µl of Smon0123 mutants were mixed with equal volume of the reservoir solution for crystallization and a crystal formed at 20 °C for 2 weeks. In the case of ligand-free Smon0123 (N-28/C-5), 21.2 mg/ml Smon0123 (N-28/C-5) was crystallized in the drop consisting of 0.2 M potassium sodium tartrate tetrahydrate, 0.1 M tri-sodium citrate dehydrate (pH 5.6), and 2 M ammonium sulfate. The 12.5 mg/ml Smon0123 (N-18/C-5) was crystallized in the drop containing 1 mM C∆0S, 0.2 M ammonium chloride, 0.1 M MES (pH 6.0), and 20% (w/v) polyethylene glycol (PEG) 6000 for the complex Smon0123 (N-18/C-5)/C∆0S; 1 mM C∆4S, 0.2 M ammonium chloride, 0.1 M HEPES (pH 7.0), and 20% (w/v) PEG 6000 for the complex Smon0123 (N-18/C-5)/C∆4S; and 1 mM C∆6S, 0.2 M potassium thiocyanate, and 20% (w/v) PEG 3350 for the complex Smon0123 (N-18/C-5)/C∆6S. Each single crystal was picked up by a nylon loop, soaked in mother liquor containing 20% ethylene glycol as a cryoprotectant, and frozen in a cold nitrogen gas stream on the beamline BL-38B1 of SPring-8 (Harima, Japan). X-ray diffraction images were collected with a MAR225HE detector (Rayonix) with synchrotron radiation at wavelength 1.00 Å. The data were indexed, integrated, and scaled using the HKL-2000 program. The structure was determined through molecular replacement method with the Molrep program in the CCP4 program package. Structure refinement was conducted using the phenix refine program in the PHENIX program package. After each refinement cycle, the model was adjusted manually with the winCoot program. R work/R free values of Smon0123 (N-18/C-5)/C∆0S, Smon0123 (N-18/C-5)/C∆4S, Smon0123 (N-18/C-5)/C∆6S, and Smon0123 (N-28/C-5) are 17.4/20.5, 19.3/21.8, 19.7/24.8, and 20.7/23.6, respectively. The torsion angles of some residues were outlier in the Ramachandran plot analysis due to those distorted β-turn structures and ambiguous electron density. Figures of protein structures were prepared using PyMOL . […]

Pipeline specifications

Software tools HKL-2000, Molrep, CCP4, PHENIX, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Bacteria, Homo sapiens, Escherichia coli, Streptobacillus moniliformis, Dipturus trachyderma
Diseases Colonic Neoplasms
Chemicals Adenosine Triphosphate, Chondroitin Sulfates