Away from the edge II: in-house Se-SAS phasing with chromium radiation.

Published

Journal Article

Recently, the demands of high-throughput macromolecular crystallography have driven continuous improvements in phasing methods, data-collection protocols and many other technologies. Single-wavelength anomalous scattering (SAS) phasing with chromium X-ray radiation opens a new possibility for phasing a protein with data collected in-house and has led to several successful examples of de novo structure solution using only weak anomalous scatterers such as sulfur. To further reduce data-collection time and make SAS phasing more robust, it is natural to combine selenomethionine-derivatized protein (SeMet protein) with Cr Kalpha radiation to take advantage of the larger anomalous scattering signal from selenium (f'' = 2.28 e(-)) compared with sulfur (f'' = 1.14 e(-)). As reported herein, the crystal structure of a putative chorismate mutase from Clostridium thermocellum was determined using Se-SAS with Cr Kalpha radiation. Each protein molecule contains eight selenomethionine residues in 148 amino-acid residues, providing a calculated Bijvoet ratio of about 3.5% at the Cr Kalpha wavelength. A single data set to 2.2 A resolution with approximately ninefold redundancy was collected using an imaging-plate detector coupled with a Cr source. Structure solution, refinement and deposition to the Protein Data Bank were performed within 9 h of the availability of the scaled diffraction data. The procedure used here is applicable to many other proteins and promises to become a routine pathway for in-house high-throughput crystallography.

Full Text

Duke Authors

Cited Authors

  • Xu, H; Yang, C; Chen, L; Kataeva, IA; Tempel, W; Lee, D; Habel, JE; Nguyen, D; Pflugrath, JW; Ferrara, JD; Arendall, WB; Richardson, JS; Richardson, DC; Liu, ZJ; Newton, MG; Rose, JP; Wang, BC

Published Date

  • July 2005

Published In

Volume / Issue

  • 61 / Pt 7

Start / End Page

  • 960 - 966

PubMed ID

  • 15983419

Pubmed Central ID

  • 15983419

Electronic International Standard Serial Number (EISSN)

  • 1399-0047

International Standard Serial Number (ISSN)

  • 0907-4449

Digital Object Identifier (DOI)

  • 10.1107/s0907444905010644

Language

  • eng