Computational protocol: Polymorphisms in the Human Inhibitory Signal-regulatory Protein α Do Not Affect Binding to Its Ligand CD47*

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Protocol publication

[…] Recombinant CD47 Ig superfamily domain and the NH2-terminal domain of SIRPαv1 (residues 1–149; accession number NP_542970) were produced in CHO Lec3.2.8.1 cells as described previously (). The proteins were purified by nickel affinity chromatography and gel-filtrated in 10 mm HEPES, pH 7, 150 mm NaCl, 0.02% NaN3. CD47 and SIRPαv1 were mixed in a 1:1 molar ratio, deglycosylated using endoglycosidase Hf, and concentrated to contain each protein at ∼620 μm. Sitting drop vapor diffusion crystallization experiments were performed using an OryxNano robot to dispense nanoscale protein precipitant drops that were equilibrated against precipitant reservoirs at 12 °C. Crystals of the CD47-SIRPαv1 complex grew from 300-nl drops containing 50% protein from 0.1 m Tris, pH 8.5, 20% w/v PEG 6000. Crystals were cryoprotected in mother liquor supplemented with 15% glycerol and flash-frozen in liquid nitrogen. Diffraction data were collected at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) at a wavelength of 0.97930 Å and were processed using Xia2 (). The structure was determined by molecular replacement using Phaser () with the CD47-SIRPαv2 structure (PDB code 2jjs) as a search model. Buccaneer () was used to autobuild followed by iterative cycles of refinement with autoBuster and manual model building in COOT (, ). […]

Pipeline specifications

Software tools xia2, Buccaneer, Coot
Application Protein structure analysis
Organisms Homo sapiens, Dipturus trachyderma
Diseases Neoplasms