Crystals were grown in very similar conditions with the PSII core complex as a starting material and diffracted to a Dibutyryl-cAMP concentration resolution of 7 and 14 Å, respectively. Materials and methods Growth and cultivation of tobacco plants The transplastomic plants of Nicotiana tabacum were created and described by Fey et al. (2008) and carry

a hexahistidine tag sequence at the 5′ end of the gene coding for the PsbE subunit. The plants were kept at a constant temperature of 25°C and at 50% relative humidity and PX-478 price grown for 10–12 weeks under a light regime of 10 h of light and 14 h of darkness per day, with a light intensity of 80–100 μmol photons/(s·m2). The plants were kept at a constant temperature of 25°C and at 50% relative humidity. PSII core complex purification Thylakoid membranes and Photosystem II core complex were purified as reported previously by Fey et al. (2008) with minor modifications. The Ni–NTA elution buffer (buffer A) had lower concentration of salt and higher concentration of the osmoprotectant betaine (10 mM MES pH 6.0, 5 mM NaCl, 1 M betaine, 5 mM CaCl2, 10 mM NaHCO3, 300 mM imidazole, 0.03% β-DDM). Size

exclusion chromatography The eluted PSII core complex was concentrated using Vivaspin 20 ultrafiltration membranes with 100 kDa cutoff until a final volume of 500 μl (at 0.5 mg/ml of chlorophylls). The protein sample was loaded on a gel filtration column (Superose 6 10/300 GL, GE Healthcare) equilibrated in buffer B (10 mM MES pH 6.0, 5 mM NaCl, 5 mM CaCl2, 10 mM NaHCO3, 0.03% β-DDM). The main peak fractions were pooled and concentrated by ultrafiltration (Vivaspin 20, 100 kDa cutoff) to a volume of 500 μl. The obtained sample was subjected to a second learn more gel filtration run and the main peak was concentrated by ultrafiltration in two steps (with Vivaspin 20, 100 kDa cutoff,

to a volume of 200 μl; and then with Vivaspin 500, 30 kDa cutoff, to a final volume of 10 μl). The chlorophyll amount in the obtained sample was determined photometrically in 80% acetone according Methocarbamol to a protocol of Porra et al. (1989) to be around 15 mg/ml. Oxygen evolution measurements Oxygen evolution was assessed with a Clark-type electrode (Hansatech, England) at 20°C in buffer B with 1 mM 2,6-dichloro-p-benzoquinone and 1 mM ferricyanide as electron acceptors in the reaction mixture. Polyacrylamide gel electrophoresis of proteins For denaturing SDS-PAGE, 10% separating Tris–tricine polyacrylamide/urea gels and 4% stacking gels were used. Samples were denatured with RotiLoad (Roth) at room temperature before loading, and after the electrophoretic separation the gels were stained with Coomassie brilliant blue (Neuhoff et al. 1988) or silver (Switzer et al. 1979). Crystallization of the PSII core complex The core complex of N. tabacum PSII was crystallized using the sitting drop vapour diffusion method at 20°C in the dark. The conditions tested for PSII crystallization were based on the ones reported by Adir (1999) and Smatanová et al. (2007).