Computational protocol: Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope

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[…] The optical density at 280 nm of each protein solution was measured before crystallization and was 0.6 and 0.4 for DENV2 sE-A259C/EDE2 A11 Fab and DENV2 sE-L107C/A313C-EDE2 A11 Fab complexes, respectively. The protein concentration estimated using theoretical extinction coefficients for the individual components, DENV2 sE-strep and EDE2 A11 Fab, was 1.03 and 1.68, respectively, without taking into account carbohydrate moieties. The protein buffer used for all the crystallization experiments was 150 mM NaCl and 15 mM Tris pH 8. Crystallization trials were performed in sitting drops of 400 nl at 18 °C. Drops were formed by mixing equal volumes of the protein and reservoir solution in the format of 96 Greiner plates, using a Mosquito robot, and monitored by a Rock-Imager. Crystals were optimized with a robotized Matrix Maker and Mosquito setups on 400 nl sitting drops, or manually in 24-well plates using 2–3 μl hanging drops at 18 °C. The crystals of DENV2 sE A259C in complex with EDE2 A11 Fab were grown in 100 mM Hepes pH 7.5, 19% PEG 6 K and 1.5% (v/v) MPD. The crystals of the complex DENV2 sE L107C/A313C with EDE2 A11 Fab were obtained in 100 mM Tris pH 8.5 and 20.7% PEG 4 K. The crystals of A259C and L107C/A313C mutants were transfered in drops containg 67% of mother liquor with respectively 16% of glycerol and 16% of ethylene glycol as cryoprotectants, before being cryo-cooled in liquid nitrogen. X-ray diffraction data were collected at beam lines PROXIMA-2 at the SOLEIL synchrotron (St Aubin, France), and ID29 at the European Synchrotron Radiation Facility (Grenoble, France). Only one crystal was used for each of the data sets. Diffraction data were processed using the XDS package and scaled with SCALA or AIMLESS in conjunction with other programs of the CCP4 suite. The high resolution limits for each structure were determined using CC1/2-based cutoffs of 0.30 (ref. ). The structures were determined by molecular replacement with PHASER using the search model DENV2 sE WT in complex with EDE2 A11 Fab (PDB 4UTB).Subsequently, careful model building with COOT was done in 2Fo–Fc electron density map (), alternating with cycles of crystallographic refinement with the programs Phenix.Refine and/or BUSTER/TNT, led to the final model. Refinement was constrained to respect non-crystallographic symmetry with TLS refinement and target restraints using higher-resolution structures of DENV2 sE (from PDB 4UTA), scFv A11 (PDB 4UT7) and constant domains of Fab A11 (from PDB 4LLD). Electron density sharpening maps were computed with COOT, and helped for the manual model building. Refined crystallographic models were analysed with MolProbity, which indicated that 99.67% and 99.82% of residues were in allowed conformations, respectively for A259C and L107C/A313C structures. The figures were prepared using the PyMOL molecular Graphics System (Schrodinger)( […]

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