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

ABSTRACT: 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 juxtamembrane 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 P212121, with unit-cell parameters a = 27.24, b = 77.64, c = 91.91 Å. Assuming the presence of a monomer in the asymmetric unit gave a crystal volume per protein weight (VM) of 1.97 Å(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.

SUBMITTER: Barak R 

PROVIDER: S-EPMC3702322 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature