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Abstract：
Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54(nrb)) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conserved Drosophila behaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible sample aggregation. Here, it is reported that L-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space group P21 with a unique axis (b) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distribution L statistic, suggesting the possibility of space group P1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 in P1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured the L-test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased sample-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed.
摘要：
非POU结构域的八聚体结合蛋白(NONO，a.k.a.p54（nrb））是核基因调控中的核心角色，具有迅速兴起的医学意义。NONO是高度保守的果蝇行为/人类剪接（DBHS）蛋白家族的成员，强制性二聚体核调节介质的动态家族。然而,与NONO同源二聚体的工作受到快速不可逆样品聚集的限制。这里,据报道，L-脯氨酸稳定纯化的NONO同源二聚体，使优质溶液小角度X射线结构测定和结晶。NONO在表观空间群P21中结晶，唯一的轴（b）为408.9µ，并有孪生的证据，如累积强度分布L统计量所示，暗示空间群P1的可能性。通过分子置换的结构解决方案显示出平行于长轴取向的六个NONO同源二聚体（或P1中的12个）的超螺旋排列，导致广泛的非晶体学对称性。进一步的分析表明，晶体不是孪生的，但是收集的数据受到高度重叠的反射，模糊了L检验。使用更高能量的X射线对新晶体进行优化数据收集，较小的光束宽度和增加的样品到检测器的距离会产生非重叠反射，分辨率为2.6µ。讨论了分析和克服这一系列实际困难并产生生物信息结构所采取的步骤。
