PHluorin‐assisted expression, purification, crystallization and X‐ray diffraction data analysis of the C‐terminal domain of the HsdR subunit of the Escherichia coli type I restriction‐modification system EcoR124I. Issue 9 (8th September 2016)

• Record Type: Journal Article
• Authors: Grinkevich, Pavel; Iermak, Iuliia; Luedtke, Nicholas A.; Mesters, Jeroen R.; Ettrich, Rüdiger; Ludwig, Jost
• Is Part Of: Acta crystallographica. Volume 72:Issue 9(2016:Sep.)
• Page Start: 672
• Page End: 676
• Publication Date: 2016-09-08
• Subjects: restriction‐modification system -- EcoR124I -- HsdR -- pHluorin -- GFP -- Escherichia coli; Crystallography -- Periodicals; Crystals -- Periodicals; 548
• DOI: 10.1107/S2053230X16011626
• Languages: English
• ISSNs: 2053-230X

Abstract

Abstract : The C‐terminal domain of the HsdR subunit of the E. coli type I restriction‐modification system EcoR124I was purified and crystallized in form of a pHluorin (GFP) fusion protein and X‐ray diffraction data were collected to a resolution of 2.45 Å. Abstract : The HsdR subunit of the type I restriction‐modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C‐terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C‐terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887–1038 and yielded crystals that belonged to the orthorhombic space group C 2221, with unit‐cell parameters a = 83.42, b = 176.58, c = 126.03 Å, α = β = γ = 90.00° and two molecules in the asymmetric unit ( V M = 2.55 Å 3 Da −1, solvent content 50.47%). X‐ray diffraction data were collected to a resolution of 2.45 Å.