Harada Ayaka, Sato Yukari, Kamimura Naofumi, Venugopalan Nagarajan, Masai Eiji, Senda Toshiya
Department of Accelerator Science, School of High Energy Accelerator Science, SOKENDAI (The Graduate University for Advanced Studies), 1-1 Oho, Tsukuba, Ibaraki 305-0031, Japan.
Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan.
Acta Crystallogr F Struct Biol Commun. 2016 Dec 1;72(Pt 12):897-902. doi: 10.1107/S2053230X16018665. Epub 2016 Nov 30.
A tetrahydrofolate-dependent O-demethylase, LigM, from Sphingobium sp. SYK-6 was crystallized by the hanging-drop vapour-diffusion method. However, the obtained P321 or P321 crystals, which diffracted to 2.5-3.3 Å resolution, were hemihedrally twinned. To overcome the twinning problem, microseeding using P321/P321 crystals as microseeds was performed with optimization of the reservoir conditions. As a result, another crystal form was obtained. The newly obtained crystal diffracted to 2.5-3.0 Å resolution and belonged to space group P222, with unit-cell parameters a = 102.0, b = 117.3, c = 128.1 Å. The P222 crystals diffracted to better than 2.0 Å resolution after optimizing the cryoconditions. Phasing using the single anomalous diffraction method was successful at 3.0 Å resolution with a Pt-derivative crystal. This experience suggested that microseeding is an effective method to overcome the twinning problem, even when twinned crystals are utilized as microseeds.
从鞘氨醇单胞菌属菌株SYK-6中分离得到一种依赖四氢叶酸的O-脱甲基酶LigM，并通过悬滴气相扩散法进行结晶。然而，所获得的衍射分辨率为2.5 - 3.3 Å的P321或P321晶体为半面孪晶。为解决孪晶问题，以P321/P321晶体作为微种子进行微种子接种，并对结晶条件进行优化。结果，获得了另一种晶型。新获得的晶体衍射分辨率为2.5 - 3.0 Å，属于空间群P222，晶胞参数a = 102.0、b = 117.3、c = 128.1 Å。优化冷冻条件后，P222晶体的衍射分辨率优于2.0 Å。使用铂衍生物晶体，通过单波长反常散射法在3.0 Å分辨率下成功进行了相位分析。该经验表明，即使将孪晶用作微种子，微种子接种也是克服孪晶问题的有效方法。
