oleracea and P. sativum PSII complexes (Adir 1999). Very similar results were obtained

for the N. tabacum PSII described here. If a single detergent was present in the drops, only spherulites could be grown. More promising crystals were grown in mixtures of α- or β-DDM with α- or β-OG (similar results were obtained if the n-HTG instead of OG anomers were used) (Table 2). The most successful combination contained α-DDM and β-OG. In these conditions, at least two types of morphologically distinguishable crystals were grown. The balance between the two crystal forms depended on the amount of the detergent mixture in the crystallization AUY-922 clinical trial drop (0.1–2%). With 0.2–0.5% (w/v) concentration of every component of the detergent mixture mainly group A crystals (Fig. 3) were formed after 7 days. Smaller group B crystals (Fig. 4) appeared later, after 12–15 days. An increase of the detergent concentration shifted the balance from group A to group B crystals. At the highest detergent concentrations, the growth Midostaurin purchase of group A crystals was completely suppressed and only group B crystals were formed. Fig. 3 Crystals of PSII core complex. a Typical morphology of crystals in the crystallization drops. b Diffraction pattern under cryogenic conditions with a limiting resolution of 7.0–7.8 Å. c SDS-PAGE analysis (Coomassie staining)

of the protein content of the crystals. Crystals were harvested from a crystallization drop, washed extensively and dissolved in loading buffer. Lane 1 was loaded with molecular marker, lane 2 with washing buffer and lane 3 with the solution

many containing the dissolved crystals. The complex was composed of the subunits CP47, CP43, PsbO, D1, D2 and PsbE. The subunit identification was based on the analyses of Barber et al. (1997) and Fey et al. (2008) Fig. 4 Crystals of CP43. a Typical morphology of crystals in the crystallization drops. b Diffraction pattern recorded at room temperature with a limiting resolution of 12–14 Å. c SDS-PAGE analysis of the protein content in the crystals. Lane 1 shows the molecular marker, lanes 2 and 3 (Coomassie and silver stained, respectively) show the protein sample obtained from the dissolved crystals after extensive washing. The observed single band was attributed to the CP43 subunit of PSII Analysis of group A crystals Crystals of group A could be routinely reproduced with a mixture of α-DDM and β-OG at a concentration 0.5% (w/v) and 50 mM of the H isomers of HT. Crystals grew in 6–8 days and reached a considerable size (maximal linear dimension 0.4–0.6 mm). Coomassie stained SDS-PAGE analysis of the protein mixture in the crystals showed a typical PSII core complex pattern plus the His–PsbE (Fig. 3). In order to cryoprotect crystals, a “mock” crystallization experiment without protein but with 17% PEG 400 or 22% glycerol in the usual crystallization buffer (1 mM CaCl2, 50 mM Bis–Tris, pH 7.0, 4% PEG 4000, 0.5% α-DDM, 0.