Nicolas Foos , Jean-Baptise Florial , Mathias Eymery , Jeremy Sinoir , Franck Felisaz , Marcus Oscarsson , Antonia Beteva , Matthew W. Bowler , Didier Nurizzo , Gergely Papp , Montserrat Soler-Lopez , Max Nanao , Shibom Basu , Andrew A. McCarthy
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引用次数: 0
摘要
系列晶体学的出现使室温 X 射线晶体结构测定技术重新焕发活力并得到普及。在生理温度下测定的晶体结构揭示了蛋白质的灵活性和动态性。此外,具有挑战性的样品(如大型复合物、膜蛋白和病毒)会形成脆弱的晶体,通常难以采集用于冷冻晶体学研究。此外,典型的系列晶体学实验需要大量微晶,这主要是通过批量结晶来实现的。许多医学相关样本是在哺乳动物细胞系中表达的,产生的蛋白质数量很少,不适合批量结晶。这就限制了系列晶体学方法的应用范围。从 96 孔结晶板直接原位采集数据不仅能确定最佳衍射结晶条件,还能在环境条件下进行结构测定。在此,我们介绍一种原位串行晶体学(iSX)方法,这种方法便于从安装在欧洲同步辐射设施微聚焦光束线 ID23-2 上可快速更换的通用板支架上的结晶板进行直接测量。我们将 iSX 方法应用于一个具有挑战性的项目,即在稳定人类细胞系中表达的治疗靶标 autotaxin,以 3.0 Å 的分辨率确定最低对称 P1 空间群的结构。我们的原位数据收集策略为结构测定提供了完整的数据集,同时筛选了各种结晶条件。我们的数据分析显示,iSX 方法在微聚焦光束线中效率很高,提高了吞吐量,并展示了如何将结晶板常规用作同步加速器串行晶体学实验中呈现样品的替代方法。
In situ serial crystallography facilitates 96-well plate structural analysis at low symmetry
The determination of a challenging structure in the P1 space group, the lowest symmetry possible, shows how our in situ serial crystallography approach expands the application of crystallization plates as a robust sample delivery method.
The advent of serial crystallography has rejuvenated and popularized room-temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g. large complexes, membrane proteins and viruses) form fragile crystals that are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallization. Many medically relevant samples are expressed in mammalian cell lines, producing a meager quantity of protein that is incompatible with batch crystallization. This can limit the scope of serial crystallography approaches. Direct in situ data collection from a 96-well crystallization plate enables not only the identification of the best diffracting crystallization condition but also the possibility for structure determination under ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallization plates mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility. We applied our iSX approach on a challenging project, autotaxin, a therapeutic target expressed in a stable human cell line, to determine the structure in the lowest-symmetry P1 space group at 3.0 Å resolution. Our in situ data collection strategy provided a complete dataset for structure determination while screening various crystallization conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallization plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons.
期刊介绍:
IUCrJ is a new fully open-access peer-reviewed journal from the International Union of Crystallography (IUCr).
The journal will publish high-profile articles on all aspects of the sciences and technologies supported by the IUCr via its commissions, including emerging fields where structural results underpin the science reported in the article. Our aim is to make IUCrJ the natural home for high-quality structural science results. Chemists, biologists, physicists and material scientists will be actively encouraged to report their structural studies in IUCrJ.