title: 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.
authors.affiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 52900 Ramat-Gan, Israel.
authors.forename: Reut
authors.initials: R
authors.lastname: Barak
authors.forename: Yarden
authors.initials: Y
authors.lastname: Opatowsky
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012645:Selenomethionine / Q000378:metabolism; D000097905:Roundabout Proteins
chemical_list: D005353:Fibronectins; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D012645:Selenomethionine; D000097905:Roundabout Proteins
keywords: Robo1; SAD; Slit; selenomethionine