TD1 sample preparation

Synthetic DNA encoding for the DARPin D1²⁸ was cloned into a pET-based expression vector (see Supplementary Table ⁵) and expressed in Escherichia coli BL21 DE3 (purchased from Stratagen). The DARPin protein was purified by Ni-affinity chromatography and size exclusion chromatography. Bovine brain tubulin was purchased from the Centro de Investigaciones Biológicas (Microtubule Stabilizing Agents Group), CSIC, Madrid, Spain. The TD1 was formed by mixing tubulin and Darpin 1 in a 1:1.1 ratio and concentrated to 20 mg ml⁻¹ before crystallization. TD1 was crystallized by hanging drop vapor diffusion at 20 °C in 20–24% PEG 3350, 0.2 M Ammonium Sulfate, 0.1 M Bis-Tris Methane pH 5.5, mixing 2 μl of the complex with 2 μl of precipitant solution. On the next day, a few TD1 crystals were observed in each drop. To reach a larger number of crystals 1 μl water was then added and the drop mixed a few times. After another night incubation at 20 °C, each crystallization drop contained numerous TD1 microcrystals.

SMX sample preparation. Individual hanging drops were pooled by pipetting into an Eppendorf tube, the glass covers were carefully washed with mother liquor. Crystals were then spun down at room-temperature with 2,000 g for 1 min. Almost all supernatant was removed and crystals were re-suspended. Crystals were then pipetted into the back of a Hamilton syringe and mixed with ~ 70% monoolein through a 400 μm coupler at room temperature until the cubic phase formed.