Crystallization screens

Purified AntR (15 mg/ml) was used for initial crystallization screens using sitting drop vapor diffusion and hanging drop methods using crystallization screens from Hampton Research, Molecular Dimensions and Emerald BioSystems, Inc. Small plate-like crystals were grown in crystal screen kit condition 41 (0.1 mM HEPES sodium, pH 7.5, 10% v/v 2-propanol, and 20% w/v polyethylene glycol 4,000) in sitting drops. Addition of a 1:1 ratio of protein:reservoir solution led to a slight precipitate that cleared by addition of 1 µl of 0.1 M TCEP. Diffraction-quality crystals were obtained with Hampton additive screen condition 47 within a week. High quality crystals were flash cooled in liquid nitrogen for data collection. X-ray diffraction data were collected at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory, Berkeley, California and the Southeast Regional Collaborative Access Team (SER-CAT) facility at the Advanced Photon Source (Sapsford et al., 2006 (link)), Argonne National Laboratory. The structures were determined by molecular replacement (McCoy, 2007 (link)). Structural refinement of each data set was performed with REFMAC5 (Vagin et al., 2004 (link)) implemented with the CCP4 suite (Winn et al., 2011 (link)). The model and electron density map were visualized using COOT software (Emsley & Cowtan, 2004 (link)).

eVP35 IID proteins were purified as described previously^{16 (link); 17 ; 33} . Crystallization screens (Hampton Research) were used to identify initial conditions using purified VP35 IID protein and further optimized using in-house reagents with 10% DMSO. After 24–48 hours, 0.4 μL of 20–50 mM compound in 100% DMSO was added to crystals and incubated at 25°C for 1–24 hours. Diffraction data were collected at the Advanced Photon Source Beamline 19ID (Argonne National Laboratory) at 100 K. Ligand bound structures were solved by isomorphous replacement, using the structure of ligand free eVP35 IID (PDB ID code 3FKE) as the search model, and refined using REFMAC5³⁴ or Phenix^{35 (link)}.

Crystallization conditions were surveyed by the hanging drop method of vapor diffusion using commercially-available crystallization screens (Hampton Research) along with various combinations of relevant ligands. Single crystals suitable for X-ray diffraction analysis were grown by mixing 2 µL dialyzed rTEV-cleaved BshA solution with 2 µL Hampton Research PEG-ion 2 #22 (0.2 M ammonium citrate pH 7.0, 20% (w/v) PEG 3,350) in the presence of 5 mM UDP-N-acetylglucosamine (Sigma) and 5 mM l-malate. Crystals grew to a maximum dimension of 0.1 × 0.2 × 0.3 mm within 1 week after microseeding from the screen. Crystals were transferred to a cryoprotectant solution consisting of 75% well solution and 25% ethylene glycol with 5 mM each of UDP-N-acetylglucosamine and l-malate and flash frozen in liquid nitrogen. The crystals belonged to the C222₁ space group with unit cell dimensions of 89.1 × 163.3 × 97.2 Å.

BioUltra PEG400 (Sigma) was aliquoted and stored at −80°C and used fresh or within a year. Sucrose and SYPRO-Orange dye were from Sigma-Aldrich Inc, and crystallization screens were from Hampton Research (Viejo CA).

Chemicals were obtained from Sigma Aldrich or Alfa Aesar. Crystallization screens were obtained from Hampton Research.