Single isomorphous replacement phasing of selenomethionine-containing proteins using UV-induced radiation damage

Santosh Panjikar, Hubert Mayerhofer, Paul A. Tucker, Jochen Mueller-Dieckmann, Daniele De Sanctis

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12 Citations (Scopus)

Abstract

The most commonly used heavy-atom derivative, selenium, requires the use of a tunable beamline to access the Se K edge for experimental phasing using anomalous diffraction methods, whereas X-ray diffraction experiments for selenium-specific ultraviolet radiation-damage-induced phasing can be per-formed on fixed-wavelength beamlines or even using in-house X-ray sources. Several nonredundant X-ray diffraction data sets were collected from three different selenomethionine (Mse) derivatized protein crystals at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation using 266 nm lasers of flux density 1.7 1015 photons s -1 mm-2 for 10-50 min. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation-damage-induced phasing (RIP) technique. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing when no tunable X-ray source is available. The behaviour of Mse derivatives of various proteins provides novel insights and an initial basis for understanding the mechanism of selenium-specific UV radiation damage.

Original languageEnglish
Pages (from-to)32-44
Number of pages13
JournalActa Crystallographica Section D: Biological Crystallography
Volume67
Issue number1
DOIs
Publication statusPublished - Jan 2011
Externally publishedYes

Keywords

  • experimental phasing
  • ID23-1
  • radiation damage
  • UV-RIP

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