Acta Crystallographica Section D

Biological Crystallography

Volume 61, Part 7 (July 2005)

research papers

Acta Cryst. (2005). D61, 960-966 [doi:10.1107/S0907444905010644]

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

H. Xu, C. Yang, L. Chen, I. A. Kataeva, W. Tempel, D. Lee, J. E. Habel, D. Nguyen, J. W. Pflugrath, J. D. Ferrara, W. B. Arendall III, J. S. Richardson, D. C. Richardson, Z.-J. Liu, M. G. Newton, J. P. Rose and B.-C. Wang

Abstract: 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 K [alpha] radiation to take advantage of the larger anomalous scattering signal from selenium ( = 2.28 e^-) compared with sulfur ( = 1.14 e^-). As reported herein, the crystal structure of a putative chorismate mutase from Clostridium thermocellum was determined using Se-SAS with Cr K [alpha] 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 K [alpha] wavelength. A single data set to 2.2 Å 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.

PDB reference: 1xho

Keywords: in-house data collection; soft X-rays; chromium rotating anode; selenomethionine; structural genomics.


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