title: Combining in situ proteolysis and mass spectrometry to crystallize Escherichia coli PgaB.
abstract: The periplasmic poly-β-1,6-N-acetyl-D-glucosamine (PNAG) de-N-acetylase PgaB from Escherichia coli was overexpressed and purified, but was recalcitrant to crystallization. Use of the in situ proteolysis technique produced crystals of PgaB, but these crystals could not be optimized for diffraction studies. By analyzing the initial crystal hits using SDS-PAGE and mass spectrometry, the boundaries of the protein species that crystallized were determined. The re-engineered protein target crystallized reproducibly without the addition of protease and with significantly increased crystal quality. Crystals of the selenomethionine-incorporated protein exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to 2.1 Å resolution.
authors: Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada.
authors: Dustin J
authors: DJ
authors: Little
authors: John C
authors: JC
authors: Whitney
authors: Howard
authors: H
authors: Robinson
authors: Patrick
authors: P
authors: Yip
authors: Mark
authors: M
authors: Nitz
authors: P Lynne
authors: PL
authors: Howell
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000378:metabolism; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D059748:Proteolysis
chemical_list: D029968:Escherichia coli Proteins; D000581:Amidohydrolases; C575184:poly-beta-1,6-N-acetyl-D-glucosamine de-N-acetylase, E coli