X-ray structure and activities of an essential Mononegavirales L-protein domain

Paesen, Guido C.; Collet, Axelle; Sallamand, Corinne; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne; Grimes, Jonathan M.

Crystallization, structure solving, refinement and validation. Crystallization was carried out by vapour diffusion at 20.5°C using 96-well sitting drop plates (Greiner) 26 . Protein pellets were dissolved in water to 5-6 mg ml À 1 , and initial crystals were obtained by equilibrating 100 nl of protein with 100 nl of reservoir solution C11 of the PGA-HT screen (Molecular Dimensions; pH 6.5) supplemented with guanidine hydrochloride (to 0.1 M), against 0.1 ml of reservoir. Glycerol was added (to 20% v/v) for cryoprotection. Diffraction data were collected at 100 K on Diamond beamlines I02, I03, I04 and I24 (Harwell, UK), and processed using the Xia2 programme suite 27 . A single-wavelength anomalous dispersion experiment allowed determination and refinement of the positions of selenium atoms, as well as calculation of the phases with autoSHARP 28 . An initial model was obtained using MR-ROSETTA 29 , enabling manual model building using COOT 30 . Refinement was performed using autoBUSTER 31 , and validation employed COOT and Molprobity 32 . Molecular replacement (using PHASER 33 ) was used to solve additional CR-VI þ structures. Refinement statistics are given in Table 1 , and a portion of the electron density map is shown in Supplementary Fig. 8 .