Crystallization of Tubulin-DARPin Complex

For crystallization, the tubulin-DARPin D1 (TD1) complex was formed by mixing the respective components in a 1:1.1 molar ratio. The TD1 complex was crystallized using EasyXtal 15-well plates by the hanging drop vapor diffusion method (drop size 2 μl, drop ratio 1:1, 8 drops per well) at a concentration of 9.8 mg ml⁻¹ at 20 °C with a precipitant solution containing 21% (w/v) polyethylene glycol (PEG) 3000, 0.2 M ammonium sulfate, and 0.1 M bis-tris methane, pH 5.5. All drops were subsequently hair-seeded with crystalline material obtained in previous crystallization trials to increase the homogeneity and density of crystals. After 48 h, crystals were washed off the plates with precipitant solution, collected in 0.6 ml tubes, and vortexed for a few seconds. This procedure induces batch crystallization within the tubes and a sedimented crystal pellet was formed after 24 h of incubation at 20 °C. Initially, crystals grew as long needles that were broken into smaller fragments of approximately 20 × 20 × 5 μm³ during the sample preparation described below.