Mock crystallization drops were equilibrated against the standard reservoir buffer for 1–2 days. The pretreatment of crystals

in the equilibrated drops significantly reduced damage (cracking) upon their transfer to the cryo-buffer. Crystals that were pretreated diffracted to a resolution of 7.0–7.8 Å at the ESRF microfocus beamline ID23-2, Grenoble (Fig. 3). Analysis of group B crystals The crystals of group B appeared hexagonal with regular or irregular shape and dimensions between 0.02 and 0.2 mm on the hexagonal face (Fig. 4). The AR-13324 purchase time of growth and crystal morphology were correlated. In the presence of a low CBL0137 price amount of detergent, group B crystals took 6–15 days to grow and were rather irregular. In the presence of a high amount of detergent (1–2% w/v), crystals took only about 3 days to appear and were more regular. The final size of group B crystals was dependent on the amount of HT (H isomers). When a lower amount of HT (25 mM) was used, crystal dimensions (across the hexagonal face) were limited to 0.01–0.05 mm. With higher amounts of HT (50–100 mM), bigger crystals with dimensions in the 0.05–0.1 mm range were obtained. The protein content of group B crystals was analyzed by SDS-PAGE followed by silver staining. Only a single band

was observed, which migrated slightly XAV-939 price faster than the 45 kDa molecular mass marker suggesting that the band represented the PSII core subunit CP43, which is known to be separable from the PSII core (Rhee et al. 1997; Büchel et al. 2000). Test exposures of the hexagonal crystals at Diamond (Didcot, UK) and at the ESRF ID23-2 (Grenoble, France) resulted in diffraction to a maximum resolution of 12–14 Å, but only for one orientation of the crystals. The recorded image showed features of diffuse scattering. We attributed this to random lattice disorder, with a short correlation length and large amplitude of displacement. Consistent with this interpretation, we observed almost no diffraction PLEKHM2 when the spindle axis was rotated by 90° (Fig. 4).

Conclusions In this work, we report the formation of two types of crystals from preparations of the PSII core complex. In the presence of a low amount of detergent mixture, crystals of the intact core complex formed first, but eventually, the CP43 core subunit alone also crystallized in the same drops. Increasing amounts of the detergents shifted the balance between the two crystal forms towards the formation of the CP43 crystals. Our findings are consistent with prior observations that the CP43 subunit can dissociate from the core complex of PSII in some conditions (Rhee et al. 1997; Büchel et al. 2000). Outlook Dehydration of membrane protein crystals has often improved diffraction quality. Therefore, controlled dehydration experiments were carried out on the crystal free mounting system (Kiefersauer et al. 2000) at Proteros (Martinsried, Germany) and directly at the ESRF, beamline ID14-2 (Grenoble, France).