蛋白晶体的微结晶和<em>在Cellulo</em>衍射
Summary
提出了使用蛋白微晶X射线晶体学的方案。两个例子体内 -grown纯化之后，或在纤维素的微晶分析进行比较。
Abstract
在许多同步加速器设备中高品质微焦距线束的出现已经允许对它们最大尺寸的小于10μm的晶体进行常规分析，这是用来代表挑战的。我们提出了通过X射线晶体学的蛋白质微晶的结构测定的两个可选工作流程，特别关注体内生长的晶体。通过超声处理从细胞中提取微晶，并通过差速离心纯化，或通过流式细胞术分析含晶体细胞进行细胞分选后的纤维素分析。任选地，将纯化的晶体或含晶体的细胞浸泡在重的原子溶液中用于实验性定相。然后将这些样品以类似的方式制备用于衍射实验，通过施加到微支架上并在液氮中快速冷却。我们简要描述和比较了分离的微晶和晶体 -使用微焦点同步加速器束线产生适合于定相，模型构建和细化的数据集。
这些工作流程的例子是用重组杆状病毒感染昆虫细胞产生的蚕豆病毒1（BmCPV1）多角体蛋白的晶体。在这个案例研究中， 纤维素分析比纯化晶体的分析更有效，并且在从表达到细化的〜8天内产生结构。
Introduction
使用X射线晶体学测定生物大分子的高分辨率结构在过去二十年中经历了稳步的发展。由非专业研究者的X射线晶体学的日益摄取体现在生命科学1许多领域这种方法的民主化。
历史上，尺寸低于〜10μm的晶体被认为是具有挑战性的，如果不可用，则用于结构测定。全球同步加速器辐射源的专用微焦距线束的可用性越来越多，技术进步（如开发微晶操作工具）已经消除了阻碍了X射线微晶体学广泛使用的这些障碍。在串行透视microcrystallography 2,3和微电子衍射4公顷进展VE表明，对于结构确定使用微米和纳米晶体的不仅是可行的，但有时也优选使用大晶体5，6，7的。
这些进展被首先施加到肽8和昆虫病毒9,10产生的天然晶体的研究。它们现在被用于各种各样的生物大分子，包括最困难的系统，如膜蛋白和大复合物11 。为了便于对这些微晶的分析，已经在内消旋 ，特别是膜蛋白12和微流体芯片13中进行了分析 。
这些新颖的方法microcrystallography的可用性已经提出使用的可能性结晶体内作为结构生物学14，15，16一个新的路线，提供一种替代传统的体外 crystallogenesis。不幸的是，即使可以产生体内晶体，仍然存在几种障碍，例如在从细胞纯化期间配体的降解或丧失，难以在同步加速器束线处的晶体的操作和可视化以及繁琐的X射线衍射实验。作为替代晶体也被直接分析细胞内不经任何纯化步骤17中，18，19。比较分析表明，这种在cellulo方法中可能比纯化晶体的分析和更高分辨率20的产量数据更有效。
这个协议是在倾向于协助研究人员进行蛋白质微结晶研究。它提供了聚焦于同步加速器束线的X射线衍射实验的样品制备和操作的方法。提出了两种选择，使用分离的晶体进行经典的微结晶法或含有细胞的细胞，通过流式细胞仪分析在纤维素分析中（ 图1 ）。
Protocol
注意：已经在许多生物体中报道了体内结晶，包括在细菌，酵母，植物，昆虫和哺乳动物中（参见参考文献21 ）。在实验室中也已经实现了重组蛋白的结晶，其使用哺乳动物细胞的瞬时转染和昆虫细胞的杆状病毒感染。已经使用根据参考文献22中的说明产生的在杆状病毒多角体启动子下克隆在重组杆状病毒中的家蚕病毒1（BmCPV1）多角体蛋白基因的?…
Representative Results
提出了使用体内微晶的结构测定的两种替代方法的概述（ 图1 ）。多面体可以通过超声处理和离心容易地纯化。由于它们的密度，它们在管的底部在可以通过移液移除的碎屑层下面形成一层（ 图3a和3b ）。然后将样品进行几轮超声波处理并洗涤，直至从颗粒的白色和白垩方面判断达到足够的纯度（ <strong class…
Discussion
该方案提供了两种分析微晶的方法，目的是便于分析过去将被忽略的非常小的晶体。
微晶纯化的关键步骤
所提出的方案已经使用以Sf9细胞表达的家蚕 CPV1多角体蛋白作为模型系统进行了优化。然而， 体内微晶显示出很大的机械阻力变化。例如，在昆虫细胞中生长的组织蛋白酶B的针状晶体是刚性的，并且对机械应力高度抗性，并且可以使用类似…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢Chan-Sien Lay提供纯化微晶的图片，Daniel Eriksson和Tom Caradoc-Davies，以支持澳大利亚同步加速器的MX2射束，以及位于蒙纳士大学FlowCore工厂的Kathryn Flanagan和Andrew Fryga，他们无价的援助
Materials
| Sf9 cells | Life Technologies | ||
| SF900-SFM insect medium | Life Technologies | ||
| 1L cell culture flask | Thermofisher Scientific | ||
| Shaking incubator for insect cell culture | Eppendorf | ||
| 50mL conical tubes | Falcon | ||
| Centrifuge with swing buckets for 50mL tubes | Eppendorf | ||
| Sonicator equiped with a 19mm probe | MSE Soniprep 150 | ||
| Glass slides | Hampton Research | ||
| Hemacytometer | Sigma-Aldrich | ||
| Propidium iodide | Thermofisher Scientific | ||
| BD Influx cell sorter | BD Biosciences | ||
| Hampton Heavy atom screens | Hampton Research | ||
| Microcentrifuge | Eppendorf | ||
| Micromesh | Mitigen | 700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies. | |
| Paper wick | Mitigen | The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used. | |
| Ethylene glycol | Sigma-Aldrich | ||
| Trypan blue | Life Technologies | ||
| MX2 microfocus beamline | Australian Synchrotron | A list of available microfocus beamlines can be found in Boudes et al. (2014) Reflections on the Many Facets of Protein Microcrystallography. Australian Journal of Chemistry 67 (12), 1793–1806, doi:10.1071/CH14455. |
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Cite This Article
Boudes, M., Garriga, D., Coulibaly, F. Microcrystallography of Protein Crystals and In Cellulo Diffraction. J. Vis. Exp. (125), e55793, doi:10.3791/55793 (2017).