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.
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 C2221, 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 (VM = 2.55 Å(3) Da(-1), solvent content 50.47%). X-ray diffraction data were collected to a resolution of 2.45 Å.
Center for Nanobiology and Structural Biology, Institute of Microbiology ASCR, v.v.i., Zamek 136, 37333 Nove Hrady, Czech Republic.
Pavel
P
Grinkevich
Center for Nanobiology and Structural Biology, Institute of Microbiology ASCR, v.v.i., Zamek 136, 37333 Nove Hrady, Czech Republic.
Iuliia
I
Iermak
Center for Nanobiology and Structural Biology, Institute of Microbiology ASCR, v.v.i., Zamek 136, 37333 Nove Hrady, Czech Republic.
Nicholas A
NA
Luedtke
Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, Lübeck, Germany.
Jeroen R
JR
Mesters
Center for Nanobiology and Structural Biology, Institute of Microbiology ASCR, v.v.i., Zamek 136, 37333 Nove Hrady, Czech Republic.
Rüdiger
R
Ettrich
Center for Nanobiology and Structural Biology, Institute of Microbiology ASCR, v.v.i., Zamek 136, 37333 Nove Hrady, Czech Republic.
Jost
J
Ludwig
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction
D029968:Escherichia coli Proteins; C485187:PHluorin; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific; C109830:HsdR protein, E coli
EcoR124I; Escherichia coli; GFP; HsdR; pHluorin; restriction-modification system