Production, biophysical characterization and crystallization of Pseudomonas putida GraA and its complexes with GraT and the graTA operator

Ariel Talavera Perez, Hedvig Tamman, Andres Ainelo, San Hadzi, Abel Garcia-Pino, Rita Horak, Albert Konijnenberg, Remy Loris

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

The graTA operon from Pseudomonas putida encodes a toxin-antitoxin module with an unusually moderate toxin. Here, the production, SAXS analysis and crystallization of the antitoxin GraA, the GraTA complex and the complex of GraA with a 33 bp operator fragment are reported. GraA forms a homodimer in solution and crystallizes in space group P2 1, with unit-cell parameters a = 66.9, b = 48.9, c = 62.7 Å, β = 92.6°. The crystals are likely to contain two GraA dimers in the asymmetric unit and diffract to 1.9 Å resolution. The GraTA complex forms a heterotetramer in solution. Crystals of the GraTA complex diffracted to 2.2 Å resolution and are most likely to contain a single heterotetrameric GraTA complex in the asymmetric unit. They belong to space group P4 1 or P4 3, with unit-cell parameters a = b = 56.0, c = 128.2 Å. The GraA-operator complex consists of a 33 bp operator region that binds two GraA dimers. It crystallizes in space group P3 1 or P3 2, with unit-cell parameters a = b = 105.6, c = 149.9 Å. These crystals diffract to 3.8 Å resolution.The antitoxin GraA from P. putida and its complexes with the toxin GraT and with the 33 bp operator of the graTA operon were crystallized.

Original languageEnglish
Pages (from-to)455-462
Number of pages8
JournalActa Crystallographica Section F - Structural Biology Communications
VolumeF73
Issue number8
DOIs
Publication statusPublished - Aug 2017

Keywords

  • Amino Acid Sequence
  • Antitoxins/chemistry
  • Bacterial Proteins/chemistry
  • Bacterial Toxins/chemistry
  • Cloning, Molecular
  • Crystallization
  • Crystallography, X-Ray
  • Escherichia coli/genetics
  • Gene Expression
  • Gene Expression Regulation, Bacterial
  • Genetic Vectors/chemistry
  • Operon
  • Protein Binding
  • Protein Multimerization
  • Pseudomonas putida/chemistry
  • Recombinant Fusion Proteins/chemistry
  • X-Ray Diffraction

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