Computational protocol: Structure of the Tandem Fibronectin Type 3 Domains of Neural Cell Adhesion Molecule

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Protocol publication

[…] NCAM 1FN3–2FN3 was concentrated to 13 mg/ml in TBS, and crystals were obtained by hanging drop vapour diffusion at room temperature using 2.2 M ammonium sulfate, 0.1 M sodium citrate, pH 5.2, 0.2 M potassium/sodium tartrate and 3–5% ethanol as precipitant. Crystals grew within 2 days and belong to space group P212121 with unit cell dimensions a = 52.77 Å, b = 71.35 Å and c = 98.22 Å. There are two 1FN3–2FN3 molecules in the asymmetric unit, resulting in a solvent content of ∼ 38%. Crystals were flash-frozen in liquid nitrogen after brief soaking in mother liquor supplemented with 20% glycerol. A crystal was soaked in mother liquor supplemented with 300 mM potassium iodide for 30 s before freezing to obtain a heavy atom derivative. Diffraction data from native and KI derivative crystals were collected at 100 K on station 14.1 at the Synchrotron Radiation Source (SRS) Daresbury and on station ID29 at the European Synchrotron Radiation Facility Grenoble, respectively. The NCAM 1FN3–2FN3 M610R mutant was concentrated to 14 mg/ml in TBS, and crystals were obtained by sitting drop vapour diffusion at room temperature using 2 M ammonium sulfate and 0.1 M sodium acetate, pH 4.6, as precipitant. Crystals grew within 3–4 days and belong to space group P212121 with unit cell dimensions a = 92.74 Å, b = 107.49 Å and c = 161.18 Å. There are six copies of mutant 1FN3–2FN3 in the asymmetric unit, resulting in a solvent content of ∼ 42%. Crystals were flash-frozen in liquid nitrogen after brief soaking in mother liquor supplemented with 20% glycerol, and diffraction data were collected at 100 K on station 10.1 at the SRS Daresbury. The diffraction data were processed with MOSFLM and programs of the CCP4 suite. The structure of NCAM 1FN3–2FN3 was solved by single-wavelength anomalous dispersion phasing of a KI-soaked crystal using SHARP (Globalphasing Ltd., Cambridge) in full automatic mode. The structure was rebuilt with O and refined with Crystallography & NMR System. The structure of the NCAM M610R mutant was solved with some difficulty by molecular replacement with PHASER, using the isolated FN3 domains of the NCAM 1FN3–2FN3 structure as search models. Data collection, phasing and refinement statistics are summarised in . The figures were made with PyMOL. […]

Pipeline specifications

Software tools iMosflm, CCP4, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens, Chrysanthemum x morifolium
Chemicals Neural Cell Adhesion Molecules