Purification, crystallization and preliminary crystallographic analysis of DR0248, an MNT-HEPN fused protein from Deinococcus radiodurans

Gaelle Pesce, Simone Pellegrino, Sean Mcsweeney, Ana Maria Goncalves, Daniele De Sanctis

Research output: Contribution to journalArticle

Abstract

DR0248 is a protein identified in the Deinococcus radiodurans (DR) genome that is predicted to encompass two domains: an N-terminal minimal nucleotidyl transferase domain (MNT) and a C-terminal higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN). These two domains, usually encoded in two ORFs, have been suggested to play the role of a toxin-antitoxin (TA) system in prokaryotes. Recombinant DR0248 was overexpressed and purified from Escherichia coli and diffraction-quality crystals were obtained in the presence of the detergent molecules dodecyldimethylamine oxide (DDAO) and octaethylene glycol monododecyl ether (C12E8), which were used as crystallization additives. Crystals grown with DDAO diffracted to a resolution of 2.24Å and belonged to space group C2221, with unit-cell parameters a = 98.4, b = 129.9, c = 59.2Å. Crystals grown with C12E8 diffracted to a resolution of 1.83Å and belonged to space group P212121, with unit-cell parameters a = 51.6, b = 87.2, c = 108.2Å. The structure was solved by multiwavelength anomalous dispersion from zinc bound to the protein using a single crystal obtained in the presence of DDAO.

Original languageEnglish
Pages (from-to)49-53
Number of pages5
JournalActa Crystallographica Section F:Structural Biology Communications
Volume71
DOIs
Publication statusPublished - 1 Jan 2015

Keywords

  • Deinococcus radiodurans
  • DR0248
  • higher eukaryotes and prokaryotes nucleotide-binding domain
  • minimal nucleotidyl transferase domain

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