Computational protocol: Structural Studies of the Spinosyn Forosaminyltransferase,SpnP

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Protocol publication

[…] Crystals of SpnP were grown using the sitting-drop vapor-diffusion method at 22 °C. The crystallization buffer consisted of 7 mM 17-pseudoaglycone 7, 46% (w/v) PEG 200, 0.6–3.0% (w/v) dextran sulfate MR 5000, 0.2 M sodium chloride, and 0.1 M phosphate citrate buffer (pH 4.24). Each drop consisted of 2 μL of the SpnP protein solution (20 mg/mL) and 1 μL of the crystallization solution with 500 μL of crystallization buffer in the reagent reservoir. Crystals were flash-frozen in liquid nitrogen prior to data collection. Crystals of SpnP complexed with TDP were obtained by supplying 20 mM TDP (final concentration) to the crystallization drop and incubating it for 1 h prior to flash-freezing. Data for SpnP and the SpnP–TDP complex were collected at Advanced Light Source beamlines 5.0.2 and 5.0.3, respectively, and processed with HKL2000. Molecular replacement was utilized to determine the unliganded SpnP structure, using the EryCIII monomer [Protein Data Bank (PDB) entry 2YJN] as the search model in Phaser., The model was initially refined with ARP/wARP and built through several rounds of refinement with Coot and Refmac.− ARP/wARP was used to model in waters, which were then evaluated manually. The TDP complex structure was determined by molecular replacement using the unliganded SpnP structure. […]

Pipeline specifications

Software tools ARP/wARP, Coot
Application Protein structure analysis
Diseases Frontotemporal Dementia