File:Controlled-In-Meso-Phase-Crystallization-–-A-Method-for-the-Structural-Investigation-of-Membrane-pone.0035458.s006.ogv

English: Time course of CIMP crystallization of bacteriorhodopsin (BR). For t = 14 hours, the experimental progress was recorded (Olympus SZX10, Olympus camera DP20) under constant cold illumination by glass fiber optics (Schott KL1500, Osram 150 W, Settings: half light, power level 1) to avoid uncontrolled changes in temperature. A brief script of what the movie shows is provided: at t = 0, swelling of 150 µg monoolein was initiated by dispensing of 450 nl BR protein solution (16 mg/ml). At t = 3 hours, 450 nl of 1∶4 dilution of reservoir solution were added to the protein well and equilibrated against undiluted reservoir solution (2.8 M Na/K phosphate, pH 5.8). Swelling continues and passes into equilibration phase. A BR protein concentration gradient from left to right can be clearly observed by the intensity of the purple color (left: high BR concentration; right: low BR concentration). At t = 6:30 hours, the microscope is refocused. At t = 7:45 hours, a local collapse of the cubic phase is observed in the centre of the well. After 10 hours, first crystals become visible on the right, the area of lowest protein concentration. At t = 10:30 hours, the movie zooms into the crystallization area. Crystals keep on growing until the end of the movie at t = 14 hours. The largest BR crystal visible in the time-lapse movie is approximately 30 µm.