使用脂质单层方法进行电子晶体学研究的样品制备
Summary
几十年来，脂质单层一直被用作形成二维（2D）蛋白质晶体的基础，用于结构研究。它们在气水界面上稳定，可作为电子成像的薄支撑材料。在这里，我们介绍了为生物研究准备脂质单层的行之有效的步骤。
Abstract
电子晶体学是高分辨率结构测定的有力工具。可溶性或膜蛋白等大分子可以在有利条件下生长成高有序的二维（2D）晶体。已种植的 2D 晶体的质量对于通过 2D 图像处理最终重建的分辨率至关重要。多年来，脂质单层已被用作支撑层，以培养外周膜蛋白和可溶性蛋白的二元结晶。该方法还可用于整体膜蛋白的二元结晶，但需要更广泛的经验调查，以确定洗涤剂和透析条件，以促进分隔到单层。在空气-水接口形成脂质单层，使极地脂质头组在水分相中保持水分，非极性乙酰链条、尾部分裂到空气中，打破表面张力，使水面变平。头部组的带电性质或独特的化学形态为溶液中的蛋白质提供了亲和力，促进二维阵列形成的结合。带有 2D 阵列的新形成的单层器可以很容易地转移到碳涂层铜网格上的电子显微镜 （EM）中，用于提升和支持晶体阵列。在这项研究中，我们描述了低温电子微观（低温-EM）成像的脂质单层方法。
Introduction
通过2D晶体或蛋白质螺旋阵列的电子衍射，在有利的情况下可以达到亚纳米分辨率1，2，3。特别感兴趣的是重建的二元膜蛋白质阵列或晶体在其近本土环境1。由于晶体充当信号放大器，可增强特定空间频率下结构因子的强度，因此电子晶体学允许探测比单粒子低温-EM尺寸较小的目标，如小分子。电子束可以通过有序的二维蛋白质阵列衍射，生成衍射模式或晶格图像，具体取决于图像平面记录在探测器4上的位置。然后，可以提取和处理衍射强度，以重建晶体的二维投影结构。电子的散射横截面比X射线大，其散射主要遵循卢瑟福模型，该模型基于电子与分子5中带电原子之间的库隆布相互作用。2D膜晶体的厚度通常小于100纳米，适合在标本6内不发生动态散射的电子传输。电子晶体学研究已被证明是探测膜蛋白和脂蛋白相互作用7、8、9、10、11、12、13、14、15、16、17的高分辨率结构信息的有力工具。
脂质单层是一个单脂层，由空气-水接口6密集包装的磷脂组成，可协助可溶性蛋白质或外周膜蛋白18的二元阵列形成。根据脂质的密度及其横向压力，脂质分子可以在空气-水接口上形成有序的二维阵列，其乙酰链延伸到空气中，亲水性头组暴露在水溶液1，6，19。脂质头组可以通过静电相互作用与蛋白质相互作用，也可以进行修改，以提供亲和力标记来结合特定的蛋白质域。例如， DOGS-NTA-Ni （1，2-二恶英-sn-甘油-3-[（N-（5-氨基-1-卡盒式）氨基酸）苏奇尼）2-尼2+）常用于形成脂质单层，将蛋白质与多组蛋白标签20，21，22结合。此外，霍乱毒素B可以结合一个特殊的五氯苯甲酸GM1在脂质单层结构研究23，24。通过将蛋白质固定在脂质头组上，脂质单层可以协助形成用于高分辨率电子晶体学研究的薄的 2D 阵列。脂质单层技术已用于蛋白质结构研究的电子晶体学， 如链球菌素2，25，附件素V26，霍乱毒素27，大肠杆菌B亚单位28，大肠杆菌RNA聚合酶25，29，30，卡盒体壳蛋白31和HIV-132和莫洛尼穆林白血病病毒的辣椒蛋白33.由于脂质单层的稳定性和化学特性，已探索了冷冻-EM成像34的样品制备应用。但是，蛋白质阵列的形成需要优化。
在这里，我们提供了关于脂质单层器用于低温-EM成像的一般准备的广泛细节，以及一些可能影响已形成单层质量的考虑。
Protocol
1. 特氟隆块准备 从耐化学PTFE（聚氟乙烯）树脂中制备特氟龙块。使用一般钻头在块上打洞，然后用 图1中标出的尺寸。 2. 单层脂质制剂 注：估计运行时间：30-45分钟 脂质库存准备 使用 8.91 mL 氯仿/甲醇、0.99 mL 甲醇和 0.01 mL 10 毫克/mL 脂质，在 9：1 （v/v） 氯仿/甲醇中准备 0.01 毫克/…
Representative Results
沉积在 EM 网格上的脂质单层可以在传输电子显微镜 （TEM） 下可视化，而不会弄脏。单层存在可以通过与光束路径中没有任何标本的区域对比差来识别。与无覆盖区域相比，脂质单层覆盖区域的局部对比度较低，因为穿过空孔的电子束没有散射，显示更亮的照明（图3）。 要筛选 2D 单层结晶的条件，使用负污渍 EM 或低温 EM 成像调查标本的图像。当舞台?…
Discussion
脂质单层是促进大型二元晶体生长的强大工具，用于生物大分子的结构研究。为了在空气-水接口成功准备一个完整的脂质单层，强烈建议在实验当天新鲜制备脂质，因为脂质乙酰链的氧化可能导致单层包装中断，并对由此产生的晶体形成产生不利影响。购买的粉末形式的脂质应使用氯仿的混合物溶解：甲醇溶剂，这是通常用来溶解磷脂35。氯仿：甲醇溶剂可以预制并储存在密封?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这份手稿的编写得到了美国陆军研究办公室（W911NF2010321）和亚利桑那州立大学启动基金对P.-L.C的部分支持。
Materials
| 14:0 PC (DMPC) | Avanti Lipids | 850345 | 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1 x 25 mg, 10 mg/mL, 2.5 mL |
| Bulb for small pipets | Fisher Scientific | 03-448-21 | |
| Chloroform | Sigma-Aldrich | C2432 | |
| Desiccator vacuum | Southern Labware | 55207 | |
| EM grids | Electron Microscopy Sciences | CF413-50 | CF-1.2/1.3-4C 1.2 µm hole, 1.3 µm space |
| Filter paper | GE Healthcare Life Sciences | 1001-090 | Diameter 90 mm |
| Glass Pasteur pipets | Fisher Scientific | 13-678-20A | |
| Hamilton syringe (25 µL) | Hamilton Company | 80465 | |
| Hamilton syringe (250 µL) | Hamilton Company | 81165 | |
| Hamilton syringe (5 µL) | Hamilton Company | 87930 | |
| Hamilton syringe (500 µL) | Hamilton Company | 203080 | |
| Methanol | Sigma-Aldrich | M1775-1GA | |
| Petri dish | VWR | 25384-342 | 100 mm × 15 mm |
| Teflon block | Grainger | 55UK05 | 60 µL wells with side injection ports, manually made |
| Tweezers | Electron Microscopy Sciences | 78325 | Various styles |
| Ultra-pure water | |||
| Ultrasonic cleaner | VWR | 97043-996 |
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Citar este artigo
Truong, C. D., Williams, D. R., Zhu, M., Wang, J. C., Chiu, P. Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies. J. Vis. Exp. (177), e63015, doi:10.3791/63015 (2021).