Expression, derivatization, crystallization and experimental phasing of an extracellular segment of the human Robo1 receptor

Robo receptors participate in the orchestration of several developmental responses, most notably axonal guidance in the central nervous system. Robo1 contains five tandem Ig-like and three fibronectin type-III (FnIII) domains in its ectodomain, followed by a single-pass transmembrane segment and an intracellular region. A human Robo1 construct that includes the two extracellular membrane-proximal fibronectin (Fn) domains and the juxtamem-brane linker was overexpressed in Escherichia coli and purified. Crystals were obtained using the vapour-diffusion method at 293 K and X-ray diffraction data were collected. Molecular-replacement attempts using related Fn domains as search models did not result in a solution. After introducing two additional methionine residues using PCR site-directed mutagenesis, selenomethionine-derivative crystals were produced. These crystals belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 27.24, b = 77.64, c = 91.91 angstrom. Assuming the presence of a monomer in the asymmetric unit gave a crystal volume per protein weight (V-M) of 1.97 angstrom(3) Da(-1) and a solvent content of 37.6%. Anisotropic diffraction data and a fragmented single-wavelength anomalous dispersion electron-density map, to which homology-modelled domains were docked, were obtained.