| Δ3-Δ2-Enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae: Molecular and structural characterization | ||
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Crystal quality was tested and the heavy atom test data sets were collected using a rotating-anode X-ray generator (Nonius) and a MAR345 image-plate detector (MAR research) at the Department of Biochemistry, University of Oulu. The data sets required for structure determination were collected at synchrotrons either in Hamburg, Germany, or in Lund, Sweden, using a MAR345 or MARCCD detector. All data sets were processed and scaled using programs DENZO and SCALEPACK (Otwinowski & Minor 1997).
Unliganded crystals were mounted in quartz capillaries and exposed to X-rays for the determination of crystal quality, the unit cell parameters and the space group. Due to the radiation sensitivity of the crystals, the conditions for cryo-cooling had to be determined. Crystals belonging to the space group P6322 with a monomer in the asymmetric unit were soaked for about 30 s in mother liquor containing an excess of ammonium sulphate (2.25 M), to prevent the crystals from solubilizing and, in addition, 20 % (v/v) ethylene glycol as cryo protectant. The crystals were then immediately flash-frozen in a stream of liquid nitrogen. The crystals were kept at 100 K during the collection of the X-ray diffraction data. The native, unliganded data set at 2.5Å resolution was collected using synchrotron radiation at beamline X11 at DESY, Hamburg.
Crystals complexed with heavy atoms were required for the determination of the crystal structure of Eci1p because attempts to use molecular replacement yielded no solutions. Heavy-atom compounds were dissolved in crystallization mother liquor in a final concentration of 1-5 mM. Crystals belonging to the space group P6322 were soaked in the heavy-atom solutions for 2-20 hours, after which X-ray diffraction data were collected in cryogenic conditions and processed as described above. The search resulted in one suitable heavy-atom derivative forming peaks in the Patterson map, potassium perrhenate (KReO4), which could be used in the MAD experiment. Prior to the MAD data collection, the crystal was soaked in 1 mM KReO4 for four hours and subsequently frozen at 100K using 20% ethylene glycol as cryo protectant.
The MAD data were collected using four wavelengths at beamline BW7A at DESY, Hamburg. The wavelengths chosen were (1) the peak of anomalous scattering of rhenium (Re), (2) the inflection point of anomalous scattering, (3) the high-energy remote and (4) the low-energy remote. The datasets were processed and scaled separately with the DENZO and SCALEPACK programs from the HKL package (Otwinowski & Minor 1977). Further data processing was done with programs of the CCP4 package (Collaborative Computational Project Number 4 1994).
Small crystals co-crystallized with octanoyl-CoA belonged to a tetragonal space group, such as P422, and diffracted at best to 3 Å resolution. There was a trimer in the asymmetric unit. X-ray diffraction data at 3.3 Å were collected at beamline 711 at Max-Lab, Lund. Before data collection, the crystal was frozen in a stream of liquid nitrogen using 20% glycerol in mother liquor as cryo protectant. The cryo solution also contained 1 mM octanoyl-CoA.