Title: Structural Insights into the Exchange Domain of Sec2p: Expression, Purification,Crystallization, and Preliminary X-Ray Diffraction Data Analysis
VOLUME: 14 ISSUE: 3
Author(s):Harindarpal S. Gill
Affiliation:Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Keywords:Pseudo-precession plot, Laue symmetry, self-rotation function, non-crystallographic symmetry, coiled-coil, nucleotide exchange factor
Abstract: Sec2p is an essential yeast gene and is part of the cell polarization process that leads to budding. The Nterminal domain of sec2p (Sec2pN) the guanine-nucleotide exchange factor for sec4p has been expressed in Escherichia coli, purified, and crystallized. Crystals belong to the space group P21 with unit cell dimensions 178.1 x 98.4 x 180.0 Å,β = 91.7°, and diffract synchrotron-generated X-rays to better than 3.6 Å resolution. Pseudo-precession plots reveal a Laue symmetry of 2/m, corresponding to the aforementioned space group, and unusual weak diffraction in the ∼5-7 Å resolution range. The Matthews number calculations for a typical crystal density suggest a range of 28 to 64 molecules per asymmetric unit. Self-rotation and native Patterson calculations demonstrate a pure helical array of protein subunits. Based on the X-ray diffraction data analysis and amino-acid sequence alignments, the paper presents a hypothetical model of the exchange domain of sec2p as a pair of coiled-coil helices that binds to sec4p and facilitates nucleotide disassociation.
