Punekar Avinash S, Selmer Maria
Department of Cell and Molecular Biology, Uppsala University, PO Box 596, 751 24 Uppsala, Sweden.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Sep;69(Pt 9):1001-3. doi: 10.1107/S1744309113020289. Epub 2013 Aug 19.
Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni(2+)-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 Å resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 Å, β = 104°. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 Å(3) Da(-1).
甲基转移酶RlmJ在大肠杆菌核糖体组装过程中利用辅因子S-腺苷甲硫氨酸对23S rRNA中的核苷酸A2030的环外氮N6进行甲基化。带有C端六组氨酸标签的RlmJ在大肠杆菌中过表达，并使用镍（2+）亲和色谱和尺寸排阻色谱法纯化得到单体。重组RlmJ采用坐滴气相扩散法结晶，并从单个无配体晶体收集到分辨率为1.85 Å的完整数据集。晶体属于空间群P2(1)，晶胞参数a = 46.9、b = 77.8、c = 82.5 Å，β = 104°。数据分析表明每个不对称单元有两个分子，马修斯系数为2.20 Å(3) Da(-1)。
