Crystallization and preliminary crystallographic analysis of a C2 protein from Arabidopsis thaliana

Abstract

An uncharacterized protein from Arabidopsis thaliana consisting of a single C2 domain (At3g17980) was cloned into the pETM11 vector and expressed in Escherichia coli, allowing purification to homogeneity in a single chromatographic step. Good-quality diffracting crystals were obtained using vapour-diffusion techniques. The crystals diffracted to 2.2 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 35.3, b = 88.9, c = 110.6 Å. A promising molecular-replacement solution has been found using the structure of the C2 domain of Munc13-C2b (PDB entry 3kwt) as the search model.

Figure 1

(a) SDS–PAGE analysis of purified His-fused AtC2 from A. thaliana. The positions of the molecular-weight markers (BioRad Laboratories Inc.) are indicated in kDa. Gels were stained using Coomassie Brilliant Blue. (b) AtC2 size-exclusion chromatography. The lines show the absorbance recorded at 280 nm. Molecular-weight markers (BioRad) are indicated in kDa. The inset corresponds to K versus log(MW). The elution position (V _e), column void volume (V ₀) and the bed volume of the column (V _t) were used to calculate K, which is defined as [(V _e − V ₀)/(V _t − V ₀)]. The elution position of recombinant AtC2 is indicated by a black triangle. (c) Monodisperity pattern obtained by dynamic light scattering (DLS) of purified AtC2.

Figure 2

AtC2 crystals grown in 0.01 M ZnCl₂, 0.1 M HEPES pH 7.0, 20%(w/v) PEG 3350 by the vapour-diffusion method.

Figure 3

Plot of the self-rotation function of an AtC2 crystal using data between 15.0–4.0 Å resolution and a 15.0 Å radius of integration in the κ = 180° section. The view is down the b axis. The highlighted peak shows a noncrystallographic twofold axis perpendicular to the crystallographic b axis.