Argc protease

Purified Δ50 TEFM (6.5 mg/ml) was treated with ArgC protease (Promega, 1:1000 w/w) and incubated for 60 min at room temperature (RT) prior to crystallization trials. Initial hits were obtained using the sitting drop vapor diffusion with a well solution containing 100 mM BIS-TRIS pH 5.5 – 6.5, 200 mM MgCl₂ and 25% PEG3350. Large, diffraction-quality crystals were obtained by the hanging drop vapor diffusion method by micro-seeding with crushed crystals into fresh drops. Crystals appeared within 24 h and grew to full size over the course of two weeks. Crystals were cryo-protected by gradually increasing the glycerol concentration to 20% (v/v) and flash frozen in liquid nitrogen.
TEFM NTD (10 mg/ml) was crystallized using the sitting drop vapor diffusion method and Crystallization Screen solution 15 (Hampton Research) containing 30% PEG4000, 0.2 M ammonium acetate, 0.1 M sodium acetate (pH 4.0) to produce large (0.05 mm × 0.05 mm × 0.8 mm) needle-like crystals. The crystals were flash-frozen using a stabilization solution containing 30% PEG4000, 15% PEG400, 0.1 M ammonium acetate and 0.05 M sodium acetate pH 4.0.

TEFM was subjected to limitied proteolysis using trypsin (Sigma, protease/TEFM ratio 1: 20,000 w/w), ArgC protease (Promega, 1:1000 w/w), LysC protease (Sigma, 1:1000 w/w) for 1 h at RT. Products of protease digestion of TEFM were dissolved in a solution containing 5% acetonitrile and 0.1% tetrafluoroacetic acid and purified using C4 ZipTip. The peptide mixtures were analyzed using a Bruker MicroFlex MALDI-TOF instrument in sinapinic acid matrix with R=5000.