Constructs of T. maritima Tm14S with altered termini (residues 107–192, 107–193, 107–194,106–191, 106–192) compared to those that produced the previous 3UR1 structure (107–191) were PCR cloned into vector pET28a (Novagen) and expressed with an N-terminal Histidine6 tag in E. coli strain BL21 (RIL DE3) (Novagen) after induction with IPTG at 18°C and overnight growth for 21 hours. The Tm14s fragments were purified first with Ni-NTA chromatography, followed by overnight thrombin digestion, and then size-exclusion chromatography (Superdex 75 Hi-load FPLC column in 50 mM NaCl, 100 mM Tris pH 7.5, 10% glycerol). T. maritima CheW and CheA Δ354 (P4P5 domain, residues 355–671) were expressed and purified as described previously34 (link).
Cubic shaped crystals (50×50×50 μm3) were grown from a mixture of 520 μM Tm14s, 457 μM CheA Δ354 and 121 μM CheW after 1 month by vapor diffusion from a 2 μl drop (1:1 mixture of protein and reservoir: 500 μl reservoir of 0.2 M sodium acetate trihydrate, 0.1 M Tris (pH 8.5), 15% w/v polyethylene glycol 4,000). Crystals with a similar shape and size as those derived from Tm14S (residues 107–191) were grown after 1 month. The new crystals (from Tm14S residues 107–192) consistently diffracted to 3.5 Å resolution. Crystals were soaked briefly in cryoprotectant that consisted of 85/15% (v/v) reservoir solution with glycerol prior to data collection in a N2 cold stream. Diffraction data were collected at 100K with synchrotron radiation at beamline A1 at the Cornell High Energy Synchrotron Source (CHESS). Selenomethionine was also incorporated into the Tm14S (residues 107–192) to aid in efforts to determine the helical registry, but unfortunately, the selenomethionine incorporated protein did not produce crystals.
Cubic shaped crystals (50×50×50 μm3) were grown from a mixture of 520 μM Tm14s, 457 μM CheA Δ354 and 121 μM CheW after 1 month by vapor diffusion from a 2 μl drop (1:1 mixture of protein and reservoir: 500 μl reservoir of 0.2 M sodium acetate trihydrate, 0.1 M Tris (pH 8.5), 15% w/v polyethylene glycol 4,000). Crystals with a similar shape and size as those derived from Tm14S (residues 107–191) were grown after 1 month. The new crystals (from Tm14S residues 107–192) consistently diffracted to 3.5 Å resolution. Crystals were soaked briefly in cryoprotectant that consisted of 85/15% (v/v) reservoir solution with glycerol prior to data collection in a N2 cold stream. Diffraction data were collected at 100K with synchrotron radiation at beamline A1 at the Cornell High Energy Synchrotron Source (CHESS). Selenomethionine was also incorporated into the Tm14S (residues 107–192) to aid in efforts to determine the helical registry, but unfortunately, the selenomethionine incorporated protein did not produce crystals.
