Structural Determination of Neuraminidase Complexes

Neuraminidase crystals were obtained by vapor diffusion from sitting drops dispensed with an Oryx 8 robot (Douglas Instruments). The drops consisted of 100 nl of protein in the absence or presence of 1 mM inhibitor (oseltamivir or zanamivir) mixed with 100 nl of reservoir solution. The reservoir solution of the I223R ligand-free crystal consisted of 20% PEG1000, 0.6 M Ammonium Phosphate and 0.1 M Sodium Acetate (pH 4.6). The reservoir solution of the I223R crystal in complex with zanamivir consisted of 18% PEG3350, 0.2 M Sodium Fluoride and 0.1 M bis-TRIS Propane buffer (pH 6.5). The reservoir solutions of the other crystals consisted of 15% PEG3350, 0.1 M bis-TRIS propane and 0.1 M sodium acetate buffer (pH 4.6). Crystals were transferred into a cryoprotectant that consisted of reservoir solution supplemented with 20% (v/v) ethylene glycol before flash freezing in liquid nitrogen. Data sets were recorded on an ADSC Q315 CCD, Pilatus 6M-F and SLS/Dectris Pilatus miniCBF detectors at the Diamond light source (Oxford, UK). Diffraction images were integrated using iMOSLFM [35] (link) or DENZO and scaled with SCALA [36] (link) or SCALEPACK for the ligand-free I223R structure. Neuraminidase structures were solved by molecular replacement with PHASER [37] (link) using the wild type structure (protein databank (PDB) identification (ID) code 2HU4) as the initial search model. Refinement was performed using Refmac5 [38] (link) or PHENIX Refine [39] (link). Manual model building was done using Coot [40] (link), structure validation was assessed with MOLPROBIDITY [41] (link) and figures were created using Pymol (http://pymol.sourceforge.net/).