Purification and crystallization of native L. reuteri GTF180-ΔN
were performed as described previously (17). Selenomethionine
(SeMet) labeled GTF180-ΔN was produced as formerly reported
(36). Both the D1025N mutant and SeMet labeled GTF180-ΔN
crystallized in the same conditions as the native protein. Crystals
of the D1025N mutant were soaked for 30 min in mother liquor
(25% ðw∕vÞ PEG 3350, 50 mM NaCl, 50 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane HCl, pH 6.0, 2 mM CaCl2
(
containing 25 mM sucrose, and cryoprotected in mother liquor
containing 35% ðw∕vÞ PEG 3350 and 25 mM sucrose. For maltose soaking studies a crystal of SeMet-GTF180-ΔN was transferred first to mother liquor supplemented with 25 mM maltose
(15 min), and then soaked for 2 h in mother liquor containing
250 mM maltose. Crystals were cryoprotected in 35% ðw∕vÞ
PEG 3350 and 250 mM maltose</quote
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posted by Scientist at 11:18 AM on December 5, 2010