Crystallization and preliminary X-ray studies of mouse centrin1

Centrins belong to a family of Ca²⁺-binding EF-hand proteins that play a fundamental role in centrosome duplication and the function of cilia. To shed light on the structure–function relationship of these proteins, mouse centrin1 has been crystallized. The mouse centrin1 has been expressed in Escherichia coli as a GST-centrin fusion protein containing a thrombin protease cleavage site between the fusion partners. Two constructs with different linking-sequence lengths were expressed and purified. Thrombin cleavage yielded functional centrin1 and N-terminally extended centrin1 containing 25 additional residues upstream of its N-terminus. Only N-terminally extended centrin1 (MW ≃ 22 240 Da) could be crystallized at room temperature, using 20–25%(w/v) PEG 1500, 5–10%(v/v) ethylene glycol and 1–2%(v/v) dioxane. Crystals were suitable for X-ray analysis, diffracting to 2.9 Å at 295 K using a rotating-anode X-ray source. They belong to space group C2, with unit-cell parameters a = 60.7, b = 59.6, c = 58.3 Å, β = 109.4°. Assuming the asymmetric cell to be occupied by one centrin1 molecule of 22.2 kDa, the unit cell contains 45% solvent with a crystal volume per protein weight, V_M, of 2.2 ų Da⁻¹.