Institut für Physikalische Biologie, Heinrich-Heine-Universitaet Duesseldorf, Universitaetsstrasse 1, 40225 Duesseldorf, Germany.
Institut für Physikalische Biologie, Heinrich-Heine-Universitaet Duesseldorf, Universitaetsstrasse 1, 40225 Duesseldorf, Germany; Institute of Biological Information Processing (IBI-7: Structural Biochemistry), Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428 Jülich, Germany.
J Struct Biol. 2020 May 1;210(2):107480. doi: 10.1016/j.jsb.2020.107480. Epub 2020 Feb 15.
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes.
核酸结构解析的主要瓶颈是结晶和相位确定。与蛋白质共结晶是克服这些挑战的直接方法。人类 RNA 结合蛋白 U1A 以前被确立为结晶模块，然而，由于缺乏紫外活性残基和在不对称单位中预定的结构，U1A 系统存在明显的局限性。在这里，我们报告了三种色氨酸 U1A 变体的晶体结构，这扩展了核酸的结晶工具箱。结构分析辅以小角 X 射线散射、NMR 光谱和光学光谱，使我们能够深入了解 U1A 变体作为核酸结晶模块的潜力。此外，我们还报告了一种通过浸泡快速有效地结晶 RNA 的方法，并提出了一种基于荧光的方法用于检测结晶中的 RNA 结合。我们的结果为 RNA 和 RNA:DNA 复合物的结晶提供了一套新的工具。
