Computational protocol: Crystal structure of mammalian acid sphingomyelinase

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Protocol publication

[…] Crystals were grown by sitting drop or hanging drop vapour diffusion at 22 °C. Protein at 10 mg ml−1 in buffer B was mixed with an equal volume of well solution. Crystals of murine ASMase with the saposin domain in an open conformation (∼200 × 25 × 25 μm in size) were obtained in 100 mM sodium MES (pH 6.5) and 1.5 M ammonium sulfate. The same protein with the saposin domain in a closed conformation was crystallized (50 × 50 × 50 μm) in 0.2 M lithium acetate and 20% PEG 3350 by first incubating it with 5 mM AbPA (Cayman Chemical) at 22 °C for 12 h. The protein was also incubated with Triton X-100 and sphingomyelin or octadecylphosphonic acid (Sigma-Aldrich) at 22 °C for 12 h, and crystals with the saposin domain in a slightly different open conformation (100 × 100 × 25 μm) were grown in 100 mM NaH2PO4, 100 mM KH2PO4, 100 mM sodium HEPES (pH 6.5) and 2 M NaCl. The same crystallization condition also yielded crystals of the isolated catalytic domain (25 × 25 × 25 μm). All crystals were briefly soaked in well solution supplemented with 20% glycerol before flash-freezing. Diffraction data were collected at 100 K on beamlines 08ID-1 equipped with a Rayonix MX300 CCD detector at a wavelength of 0.97949 Å (beam size of 130 × 30 μm), or 08B1-1 with a Rayonix MX300HE CCD detector at 1.28154 Å (230 × 195 μm) in the case of the AbPA-bound crystal, at the Canadian Macromolecular Crystallography Facility, Canadian Light Source. Data collection parameters were as follows: for the open conformation crystal, 1 s 0.45° images covering 119.7°; for the AbPA-bound crystal, 15 s 0.5° images covering 270°; for the open conformation crystal in the presence of lipid, 1 s 0.25° images covering 180°; for the catalytic domain crystal, 2 s 0.85° images covering 378.25°. Data was processed with HKL2000 (ref. ) or XDS. [...] All structures of ASMase were solved with the Phaser/Phenix molecular replacement package using a 1.4-Å resolution experimentally determined structure of the ASMase paralogue, SMPDL3A lacking the saposin domain (PDB code 5FC1). After rebuilding in Coot () and refinement in Phenix using metal coordination restraints but no non-crystallographic symmetry restraints, the saposin domain could be located and built from difference electron density. The crystal of ASMase bound to octadecylphosphonic acid had a twin fraction of 50% and was refined with the twin law h,-k,-l. Structural images were generated with PyMOL (The PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC). […]

Pipeline specifications

Software tools XDS, PHENIX, Coot, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens