Structural History of Human SRGAP2 Proteins

This is the author accepted manuscript. The final version is available from Oxford University Press via the DOI in this record.We thank Adam Frost and Eckart Gundelfinger for valuable advice on the manuscript, Michaela Vogel, Lada Gevorkyan-Airapetov, Rinat Vasserman and Tomer Orevi for technical assistance, and Hadar Amartely and Mario Lebendiker for help with SEC-MALS experiments and analysis. Thanks to the staff of beamlines ID14, ID23, and ID29 of ESRF, and the staff of BESSY II BL14.1. This work was supported by funds from the ISF (Grants no. 182/10 and 1425/15 to Y.O.) and BSF (Grant no. 2013310, to Y.O. and Adam Frost) as well as by the DFG grants QU116/6-2 to B.Q. and KE685/4-2 to M.M.K

Expression, purification and crystallization of the phosphate binding PstS protein from Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) infections pose a serious threat to human health. PA is a leading cause of fatal lung infections in cystic fibrosis and immune-suppressed patients, of sepsis in burn victims and of nosocomial infections. An important element in PA virulence is its ability to establish biofilms that evade suppression by the host’s immune system and antibiotics. PstS, a periplasmic subunit of the Pst phosphate-transport system of PA, plays a critical role in the establishment of biofilms. In some drug-resistant PA strains, PstS is secreted in large quantities from the bacteria, where it participates in the assembly of adhesion fibres that enhance bacterial virulence. In order to understand the dual function of PstS in biofilm formation and phosphate transport, the crystal structure of PA PstS was determined. Here, the overexpression in Escherichia coli and purification of PA PstS in the presence of phosphate are described. Two crystal forms were obtained using the vapour-diffusion method at 20°C and X-ray diffraction data were collected. The first crystal form belonged to the centred orthorhombic space group C222(1), with unit-cell parameters a = 67.5, b = 151.3, c = 108.9 Å. Assuming the presence of a dimer in the asymmetric unit gives a crystal volume per protein weight (V (M)) of 2.09 Å(3) Da(−1) and a solvent content of 41%. The second crystal form belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 35.4, b = 148.3, c = 216.7 Å. Assuming the presence of a tetramer in the asymmetric unit gives a crystal volume per protein weight (V (M)) of 2.14 Å(3) Da(−1) and a solvent content of 42.65%. A pseudo-translational symmetry is present in the P2(1)2(1)2(1) crystal form which is consistent with a filamentous arrangement of PstS in the crystal lattice