Open AccessCrystallization and preliminary X‐ray studies of mouse centrin1
Author(s) -
Park Jung Hee,
Krauss Norbert,
Pulvermüller Alexander,
Scheerer Patrick,
Höhne Wolfgang,
Giessl Andreas,
Wolfrum Uwe,
Hofmann Klaus Peter,
Ernst Oliver Peter,
Choe HuiWoog
Publication year - 2005
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309105011462
Subject(s) - crystallization , materials science , x ray , crystallography , chemistry , thermodynamics , physics , optics
Centrins belong to a family of Ca 2+ ‐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 C 2, 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 Å 3 Da −1 .