Crystallization of the Val264Gly mutant DPAGT1 with UDP-GlcNAc and Tunicamycin
To obtain a co-structure of DPAGT1 with UDP-GlcNAc, Val264Gly DPAGT1 was incubated with 10 mM UDP-GlcNAc for 1 hour at 4 °C before setting up crystallization with seeds at 20 °C in reservoir solution containing 32.5%–38% (v/v) PEG 300, 50 mM NaCl, 0.1 M bicine, pH 9.0. To obtain a co-structure of DPAGT1 with tunicamycin, Val264Gly DPAGT1 harvested after SEC was supplemented with 0.1 mM tunicamycin and incubated for 1 hour at 4 °C before concentrating. The concentrated protein was crystallized at 4 °C in sitting drop crystallization trials with reservoir solution containing 36% (v/v) PEG 200, 50 mM sodium chloride, 0.1 M bicine, pH 8.5.