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[…] velength anomalous dispersion (MAD) experiments were grown in 26% PEG 4000, 0.1 M Tris (pH 8.0) and 0.5 M sodium acetate. They were cryoprotected by transfer into 25% glycerol, 34% PEG 4000, 0.1 M Tris (pH 8.0), 0.5 M sodium acetate, 200 mM ascorbate and 0.5 mM β-mercaptoethanol and flash-frozen in liquid nitrogen. Diffraction data were collected to 3.0 Å at 100 K on beamline BM14 at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). Native data sets were also collected at 100 K on beamline I02 at the Diamond Light Source using crystals grown in 30% PEG 4000, 0.1 M Tris (pH 8.0) and 0.35 M sodium acetate., Native and MAD data were processed using MOSFLM and SCALA from the CCP4 suite and were consistent with a primitive tetragonal lattice. Systematic absences in the 00l and h00, 0l0 reflections indicated that the crystals belong to space group P43212. The structure was determined using MAD phasing with data from a selenium derivative. The asymmetric unit of the crystal contained two molecules, and 18 Se sites were found and refined using autoSHARP. MAD phases, following density modification, were used to calculate an initial map that was used for model building in Coot. The structure was refined against native data (100° of data at 3.2 Å) using TLS refinement in Refmac5 with 5% reflections kept excluded for Rfree evaluation. Data processing and structure refinement statistics are outlined in ., Measurements were performed on a Biacore 2000 instrument with a constant flow rate of 30 μl/min. Placental CSPG was obtained from MR4 (deposited by C. Gowda) and coupled to the surface of a CM5 chip (Biacore) using the amine coupling protocol as recommended by the manufacturer. Briefly, channels 1 and 2 were treated with a 1:1 mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (Biacore). Placental CSPG was dissolved to a concentration of 100 μg/ml in 10 mM sodium acetate (pH 5.0) and flowed through channel 2 until the response […]

Pipeline specifications

Software tools CCP4, Coot, REFMAC5