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Calcium ions play an important regulatory role in eukaryotes. However, the regulatory roles of Ca2+ in prokaryotes are poorly understood. CalD, an 18 kDa calcium-binding protein from the model actinomycete Streptomyces coelicolor A3(2), was purified and crystallized for structure determination by X-ray crystallography. Crystals of CalD that were suitable for X-ray diffraction were obtained using the hanging-drop vapour-diffusion method and diffraction data were collected in-house to 1.56 Å resolution. The crystals belonged to space group P212121, with unit-cell parameters a = 32.9, b = 51.0, c = 87.0 Å, α = β = γ = 90.0°. There is one protein molecule per asymmetric unit.
The important and diverse regulatory roles of Ca2+ in eukaryotic cells are mediated by a large family of EF-hand calcium-binding proteins (CaBPs; Clapham, 1995 ; Strynadka & James, 1989 ). These helix–loop–helix EF-hand-containing proteins (Babu et al., 1988 ) can be divided into two groups according to the function induced by Ca2+: calcium sensors convey the Ca2+ signal through an ion-induced conformational change for the recognition of target molecules (Ikura et al., 1992 ), while calcium buffers interact with the metal ions for transportation roles in eukaryotes that are less sensitive to Ca2+ loading (Ikura, 1996 ).
In contrast to eukaryotes, calcium-mediated regulatory roles are less evident in prokaryotes, despite accumulated evidence that Ca2+ is involved in a myriad of physiologically important bacterial activities, including cell division (Yu & Margolin, 1997 ), chemotaxis (Tisa & Adler, 1995 ; Watkins et al., 1995 ) and cell differentiation (Norris et al., 1991 ; Michiels et al., 2002 ; Yonekawa et al., 2001 ). Calerythrin, a well studied CaBP from Saccharopolyspora erythraea, was the first prokaryotic calcium-binding protein identified to contain four canonical calcium-binding EF-hand motifs (Leadlay et al., 1984 ; Swan et al., 1987 ) and was proposed to function as a Ca2+ buffer rather than as a Ca2+ sensor (Cox & Bairoch, 1988 ).
It has recently been reported that the CaBP from Streptomyces coelicolor, a model actinomycete responsible for producing most natural antibiotics used in medicine, is involved in the regulation of spore germination and aerial hypha formation (Wang et al., 2008 ). Cell-differentiation processes have been established to be calcium-dependent and calcium-mediated in actinomycetes (Natsume et al., 1989 ; Natsume & Marumo, 1992 ). The preliminary X-ray diffraction analysis of an 18 kDa calcium-binding protein from S. coelicolor A3(2) named CalD (SCO4411) offers a fundamental structural basis for understanding the possible regulatory roles of Ca2+ in prokaryotes. The low sequence similarity (less than 28%) between CalD and other prokaryotic EF-hand proteins (Bylsma et al., 1992 ; Michiels et al., 2002 ; Swan et al., 1987 ; Tossavainen et al., 2003 ; Yonekawa et al., 2001 ) suggests that it has a potentially distinct and significant function in physiological processes. Here, we report the expression, purification and crystallization of CalD. Preliminary crystallographic analysis shows that CalD is a stable monomer and is suitable for X-ray crystallographic studies.
We obtained the original pET-44b-CalD plasmid used to express the CalD gene (SCO4411) from Shenglan Wang (Institute of Microbiology, Chinese Academy of Sciences) and subcloned the full-length CalD gene (residues 1–170) into the bacterial expression vector pGEX-6p-1 (GE Healthcare) with BamHI and EcoRI restriction sites. The new recombinant plasmid pGEX-6p-1-CalD, which introduced a N-terminal GST tag for protein purification by glutathione S-transferase affinity chromatography, was transformed into Escherichia coli strain BL21 (DE3).
Cells were grown in Luria–Bertani (LB) medium with 100 µg ml−1 ampicillin at 310 K and were induced with 0.2 mM IPTG at an OD600 of ~0.6 for functional expression. After growth for a further 4 h, the cells were harvested by centrifugation. After resuspension in 40 ml 1×PBS (phosphate-buffered saline; 10 mM sodium phosphate pH 7.4, 150 mM NaCl), cells were lysed by sonication on ice (medium power output). The debris was removed by centrifugation (30 min at 27 000g, 277 K) and the supernatant was loaded onto a glutathione column filled with Glutathione Sepharose 4B (GE Healthcare) equilibrated with 1×PBS. The GST-tagged protein was eluted with 300 mM NaCl in 1×PBS buffer and cleaved with 200 µg GST-rhinovirus 3C protease at 277 K overnight, which left five residues (GPLGS) at the N-terminus before the CalD protein. Fractions containing CalD lacking the GST tag were pooled and desalted by ultrafiltration for further purification using Resource Q anion-exchange chromatography columns (Amersham-Pharmacia Biotech) pre-equilibrated with 20 mM HEPES pH 7.5. The protein was washed with five column volumes of 20 mM HEPES pH 7.5 and eluted with a linear 0–1.0 M gradient of NaCl in 20 mM HEPES pH 7.5. The flowthrough containing CalD fractions with A 280/A 254 > 1.25 was collected and concentrated. The target proteins were then further applied onto a Superdex 75 gel-filtration column (Pharmacia) pre-equilibrated and eluted with 20 mM HEPES pH 7.5, 150 mM NaCl. Fractions containing CalD (tested by SDS–PAGE) were collected and concentrated to 20 mg ml−1 by ultrafiltration (10 kDa molecular-weight cutoff). The purified CalD with high purity and homogeneity was analyzed by 15% SDS–PAGE (Fig. 1 ) and stored in 20 mM HEPES pH 7.5.
Crystallization trials were carried out in custom-made 16-well plates at 291 K using the hanging-drop vapour-diffusion method. Initial crystallization screening was carried out with Crystal Screens I and II (Hampton Research). A 1 µl droplet consisting of 20 mg ml−1 protein in 20 mM HEPES pH 7.5 was mixed with an equal volume of reservoir solution and equilibrated against 200 µl reservoir solution in a sealed chamber. Initial needle-like crystals of CalD appeared in Crystal Screen I condition No. 45 (18% PEG 8000, 0.1 M sodium cacodylate pH 6.5, 0.2 M zinc acetate) after 1 d and this condition was used for further optimization by variation of the precipitant concentration, buffer pH and protein concentration. Following optimization, thin tablet-like CalD crystals which diffracted well were obtained using the following conditions: 14%(w/v) PEG 8000, 0.1 M sodium cacodylate pH 6.5, 0.2 M zinc acetate (Fig. 2 ).
A single CalD crystal was harvested using a nylon loop (Hampton Research), flash-cooled to 100 K in a nitrogen-gas stream and used for data collection. A cryo-solvent was not used in the flash-cooling of the CalD crystals, since the precipitant in the crystallization solution was able to serve as a cryo-solvent. Complete diffraction data were collected at 100 K with 1° oscillation per image on an R-AXIS IV++ image-plate detector using an in-house Rigaku MicroMax-007 rotating-anode X-ray generator (Fig. 3 ). The data were indexed and scaled using the programs DENZO and SCALEPACK from the HKL-2000 package (Table 1 ).
The initial needle-like crystals exhibited high mosaicity and were not good enough for data collection. Following optimization of the initial conditions, plate-like CalD crystals which diffracted well and were suitable for data collection were obtained by reducing the concentration of the precipitant and lowering the protein concentration to 10 mg ml−1. Diffraction data were collected in-house from the plate-like crystals to 1.56 Å resolution (Fig. 3 , Table 1 ). The crystals belonged to space group P212121, with unit-cell parameters a = 32.9, b = 51.0, c = 87.0 Å, α = β = γ = 90.0° (Table 1 ). Initial analysis of the crystal solvent content using the Matthews coefficient (Matthews, 1968 ) suggested that the asymmetric unit contains one molecule with 39.4% solvent content (Matthews coefficient 2.03 Å3 Da−1). Attempts at MAD phasing based on selenomethionine substitution are in progress.
This project was supported by funds from the National Natural Science Foundation of China.