Figure 1.
Monoolein/water self-organization into mesophase: time course of mesophase formation.
900 nl water was added to a protein well of a crystallization micro plate coated with 132 µg monoolein (MO). The optical properties of the forming phases were examined under a polarization microscope over time. A, t = 1 min, B, t = 5 min C, t = 21 min, D, t = 22 min, E, t = 39 min Note: Arrows indicate the lower boundary of the forming isotropic meso-phase.
Figure 2.
The monoolein/water isotherm at 22°C.
With increasing water content, the layered lamellar phases (Lc, Lα), cubic phases (Ia3d, Pn3m) and the phase Pn3m + water are formed [5]. The transition from lamellar phase to cubic phase can be monitored by the optical property of the phase (loss of birefringence, see Figure 1). The depiction of the phases has been adapted from M. Caffrey [33]. The water content required for the formation of the individual phases can be targeted by vapor diffusion and addition of diluted screen solution as indicated (e.g. 1/1 means undiluted and 1/4 means the dilution of screen solution by a factor of 4).
Figure 3.
The effect of dilution for different partitioning constants K.
A, Fraction of total protein incorporated into lipidic phase calculated for different partitioning constants as a function of water content (upper abscissa). B, Remaining concentration of protein in the aqueous phase (supernatant including the water content of the mesophase) relative to sample concentration calculated for different partitioning constants as a function of water content (upper abscissa). The lower abscissa gives the dilutions of the screening solution added to perform the standard experiment targeting the hydration level indicated on the upper abscissa. Dilution factor 1 refers to undiluted screening solution added, factor 2 refers to a 1∶2 diluted screening solution etc. Standard experiment: 132 µg monoolein plus 450 nl protein solution (sample) plus 450 nl undiluted or diluted screening solution to be equilibrated against undiluted screening solution in the well reservoir. Calculations are based on a partitioning model, where the protein is assumed to be monomeric in both phases with K = Clip/Caq, ,where C (molality) refers to protein concentration in the lipidic or aqueous phase. Courses of lipid-incorporated and protein remaining in the aqueous phases in A and B are depicted for K values between 0 and 125 as indicated in the insets.
Figure 4.
CIMP crystallization of BR (H. salinarum).
A, Crystals from high excess water crystallization condition after 2 weeks (expected hydration level 75%). Ammonium sulfate was used as precipitant. Crystal size is approximately 11 µm. B, Crystals from excess water crystallization condition after 5 days (expected hydration level 60%). Na/K phosphate was used as precipitant. Crystal size is approximately 140 µm. C, Crystals from cubic phase crystallization conditions after 10 weeks (expected hydration level 43%). Na/K phosphate was used as precipitant. Crystal size is approximately 100 µm. D, Crystals from cubic/lamellar phase crystallization conditions after 14 weeks (expected hydration level 30%). Na/K phosphate was used as precipitant. Crystal size is approximately 200 µm.
Figure 5.
CIMP crystallization applied to Sensory Rhodopsin II (H. salinarum).
SRII crystals from cubic phase using the precipitant ammonium sulfate ((NH4)2SO4) are shown. A, First hit after 12 weeks incubation time, expected final (initial) hydration level was 43% (87%). 4.0 M (NH4)2SO4 was used as precipitant. Crystal size is approximately 6 µm. B, First diffracting crystal obtained after 3 weeks incubation time, expected final (initial) hydration level was 43% (87%). 3.3 M (NH4)2SO4, 50 mM malonate was used as precipitant. Crystal size is approximately 140 µm. C, Optimized crystallization condition, expected final (initial) hydration level was 30% (87%). 3.3 M (NH4)2SO4, 600 mM malonate was used as precipitant. D, Optimized crystallization condition, expected final (initial) hydration level was 30% (87%). 3.3 M (NH4)2SO4, 50 mM malonate was used as precipitant. Crystal size is approximately 180 µm.