澳大利亚同步加速器串行晶体测量的利皮迪科注射协议
Summary
该协议的目的是演示如何准备串行晶体学样本，以便于最近在澳大利亚同步加速器上委托的高粘度喷油器Lipidico上收集数据。
Abstract
澳大利亚同步加速器公司已开发出一种用于进行连续晶体测量的设施。该设施采用了专用的高粘性喷油器Lipidico，作为大分子晶体学 （MX2） 光束线的一部分，可在室温下测量大量小晶体。该技术的目标是使晶体能够种植/转移到玻璃注射器中，直接用于喷油器的串行晶体成像数据收集。此喷油器的优点包括能够快速响应流速变化而不中断流。这种高粘度喷油器 （HVI） 存在几个限制，其中包括对允许的样品粘度限制为 +gt;10 Pa.s。流稳定性也可能是一个问题，具体取决于样本的特定属性。此处介绍了如何设置样品和操作喷油器以进行澳大利亚同步加速器的串行晶体学测量的详细协议。该方法演示了样品的准备，包括将酶晶体转移到高粘度介质（硅胶油脂）中，以及喷油器在 MX2 中用于数据收集的操作。
Introduction
串行晶体学 （SX） 是一种最初在 X 射线自由电子激光器 （XFELs）1、2、3、4的背景下开发的技术。虽然固定目标方法可用于SX 5、6、7，但通常情况下，喷油器系统用于将晶体连续输送到X射线束。由于它结合了大量晶体的数据，SX避免了在实验过程中需要任何晶体对齐，并使数据能够在室温8，9时收集。在合适的喷油器的帮助下，晶体逐一流入X射线交互区，并在区域探测器9、10上收集由此产生的衍射数据。迄今为止，SX已经成功地解决了一些蛋白质结构1，11，12，13，包括晶体太小，无法测量使用传统的晶体学。它还利用XFEL的股秒脉冲持续时间，为时间解决的分子动力学提供了新的见解。通过使用光学激光源启动泵探针反应，对光系统II14、15、光活性黄蛋白16、17、细胞色素C氧化酶18以及细菌霍多普辛19、20、21进行了深入研究。这些研究已经探索了光激活后发生的电子转移动力学，证明了序列晶体学在理解时间解决的生物过程方面的巨大潜力。
连续晶体学的发展也越来越普遍同步加速器来源9，12，20，22，23，24。基于同步加速器的 SX 允许使用适当的喷油器系统在室温下有效地测量大量单个晶体。因此，这种方法适用于较小的晶体，因此，除了需要快速帧速探测器来收集数据外，还需要微聚焦光束。与传统的晶体学相比，SX 不涉及 X 射线束中单个晶体的安装和对齐。由于大量单个晶体的数据被合并，与传统晶体学相比，每个晶体接收的辐射剂量可以大大降低。同步加速器 SX 也可以应用于时间解决反应的研究，甚至到毫秒的机制，只要有足够高帧速率的探测器（例如，100 Hz 或更多）。在同步加速器上已经进行了几次连续晶体学实验，这些实验使用喷油器，这些喷油器最初是在XFEL源20、22、23号开发的。两种最常见的喷油器类型是气体动态虚拟喷嘴（GDVN）25和高粘性喷油器（HVI）9，24，26，27，28。GDVN 是注射低粘度液体样品的理想之选，但需要高流速才能实现稳定的流，这反过来又会导致高样品消耗率。相比之下，HVI 的适用于高粘度样品，从而以更低的流速生成稳定的流，从而降低样品消耗。因此，HVI喷油器倾向于在粘性载体更可取的情况下交付样品（例如，基于脂质的膜蛋白）和/或大量样品不可用。SX喷油器的使用通常具有挑战性，需要广泛的操作培训。它们还涉及冗长的样品传输协议，因为样品需要加载到专门的储层中，这通常与样品在”死体积”中丢失或连接中泄漏有关的风险很高。因此，在样品到达 X 射线束之前，优化喷油器设计以减轻任何损失是可取的。
最近，第一个SX结果使用利皮迪科23与酶靶点，使用Eiger 16M探测器发表。这种喷油器设计通过最大限度地减少从初始结晶到将晶体转移到喷油器中，然后将样品输送到 X 射线束中所涉及的步骤数量，从而限制样品浪费。本手稿描述并演示了从样品准备开始、进入注射过程，最后使用同一结晶容器收集数据的样品传输过程。还描述了喷油器的操作。
Protocol
Representative Results
Discussion
开发了一种替代的 HVI，非常适合在同步加速器源上进行 SX 实验。它比现有的高等健康高等疾病有两个关键优势。首先，在光束线上安装很容易，允许在传统晶体学和 SX 之间快速切换，在 MX2 上安装和对齐只需约 30 分钟。其次，用于种植晶体的样品注射器可直接用作注射液罐，从而限制样品转移过程中的浪费。已描述和演示更改样本的协议。与其他高射电相比，该设计无需复杂的气流来控制喷气?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了澳大利亚高级分子成像研究中心（CE140100011）（http://www.imagingcoe.org/）的支持。这项研究部分使用澳大利亚同步加速器的MX2光束线进行，这是ANSTO的一部分，并利用了澳大利亚癌症研究基金会（ACRF）探测器。
Materials
| Hen eggwhite lysozyme | Sigma-Aldrich | L6876 | Used to grow crystals for testing the injector and the crystals are transferred into silicon grease. https://www.sigmaaldrich.com/ |
| High vacuum silicon grease | Dow Corning | Z273554-1EA | Used for testing of injector. https://www.sigmaaldrich.com/ |
| Injector needle (108 µm ID) | Hamilton | part No: 7803-05 | www.hamiltoncompany.com |
| Glass gas-tight syringes, 100 µl | Hamilton | part no: 7656-01 | Syringes used for sample injection. www.hamiltoncompany.com |
| LCP syringe coupler | Formulatrix | 209526 | Syringe coupler to mix the samples |
| Lipidico injector | La Trobe Univerity/ANSTO | This is a specific piece of equipment that can be accessed through La Trobe University / ANSTO Australian Synchrotron Facility |
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