Crystallographic studies of the structured core domain of Knr4 from Saccharomyces cerevisiae

Abstract

The potentially structured core domain of the intrinsically disordered protein Knr4 from Saccharomyces cerevisiae, comprising residues 80-340, was expressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. Selenomethionine-containing (SeMet) protein was also purified and crystallized. Crystals of both proteins belonged to space group P6522, with unit-cell parameters a = b = 112.44, c = 265.21 Å for the native protein and a = b = 112.49, c = 262.21 Å for the SeMet protein, and diffracted to 3.50 and 3.60 Å resolution, respectively. There are two molecules in the asymmetric unit related by a twofold axis. The anomalous signal of selenium was recorded and yielded an electron-density map of sufficient quality to allow the identification of secondary-structure elements.

Keywords: Saccharomyces cerevisiae; cell-wall biogenesis regulation; hub proteins; intrinsically disordered proteins.

Figure 1

Crystals of native Knr4(80–340) (a) under visible light and (b) under UV light. (c) Crystal of SeMet-Knr4(80–340). The bar corresponds to 200 µm.

Figure 2

Fluorescence spectrum collected from SeMet-Knr4(80–340) crystals on beamline ID29 at the ESRF (f′′, red). The Kramers–Kronig transformation (f′) is represented in green.

Figure 3

Stereoview of the electron-density map obtained after SAD phasing with SHELXC/D/E and further density modification with DM. The electron-density map is displayed as a grey mesh contoured at 1σ. Constructed α-helices and β-strands are displayed as polyalanines.