It looks like you're using an Ad Blocker.
Please white-list or disable AboveTopSecret.com in your ad-blocking tool.
Some features of ATS will be disabled while you continue to use an ad-blocker.
2. Materials and methods
2.1. Crystal isolation from in vivo conditions
Crystals were extracted from D. punctata embryo midguts. The cockroaches, which were fed Lab Chow (Purina, St Louis, Missouri, USA) and water, were maintained at an ambient temperature of 27°C, with a light and dark cycle of 12 h each. 12 fertilized eggs are deposited in the brood sac of 7–8-day-old mated females. To obtain crystals, embryos were gently extruded from the brood sac of a 54-day-old female. The midgut was isolated from each embryo by cutting off the head and the end of the abdomen, allowing the midgut to be extruded into insect Ringer's solution. Supplementary Movie S1 shows how a cut made in the midgut allows its contents to be extruded by the contraction of muscles in the midgut wall. Crystals were collected in a Pasteur pipette and transferred to fresh sterile water, in which they are insoluble. Prior to X-ray diffraction experiments, crystals were cryoprotected in 20% glycerol and flash-cooled in liquid N2.
2.2. Crystallographic data-collection procedure for high-resolution crystals
Data to 1.20 Å resolution were measured using a MAR CCD detector on beamline PXII at the Swiss Light Source (SLS), Villigen, Switzerland at a wavelength of 0.8349 Å (Pohl et al., 2006). The sample-to-detector distance was set to 100 mm. All data collections were performed at cryo-temperatures using a 70 K nitrogen stream. Individual data sets were reduced with the d*TREK software (Pflugrath, 1999).
2.3. Recrystallization and data collection of solubilized protein
Lili-Mip crystals obtained in vivo were solubilized in 50 mM sodium acetate pH 5.0. Size-exclusion chromatography was carried out on the solubilized protein using a Superdex 200 prep-grade column. The protein eluted as a homogenous and monodisperse fraction at 95.5 ml and was used for crystallization. Based on the Bio-Rad Gel Filtration Standard (Bio-Rad catalogue No. 151-1901), the Lili-Mip protein was calculated to elute as a monomer with a molecular weight of about 24 kDa. Purified Lili-Mip was crystallized in 25% PEG 10 000 at a concentration of 2 mg ml−1 and a temperature of 293 K. The high PEG concentration in the crystallization condition served as the cryoprotectant and hence additional PEG or glycerol were not added. The sizes of the recrystallized and the in vivo grown crystals were similar. The size of the crystal used for data collection was about 15 × 20 µm. X-ray diffraction data for these crystals was collected on the PROXIMA-1 beamline at the SOLEIL synchrotron, France, at a wavelength of 0.97857 Å. The sample-to-detector distance was set to 270.6 mm. All data collections were performed at cryotemperature using a 100 K nitrogen stream.