我们感谢许多钻石光源科学家和支持团队成员，他们为 VMXi 光束线的设计、建造和运营做出了贡献。我们感谢光束线用户，他们后来为结晶和数据收集管道的开发贡献了想法。哈韦尔的结晶设施得到了钻石光源有限公司、罗莎琳德富兰克林研究所和医学研究委员会的支持。

自动蛋白质结晶和实时数据收集

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作者声明没有利益冲突。

我们已经描述了从蛋白质样品到达CF到用户下载最终数据以供进一步应用的完整过程。关键步骤是生产高质量的蛋白质样品和适当的晶体筛选，要么使用商业稀疏基质筛选，要么使用基于既定条件的优化筛选。这个过程可以在CF中进行，或者用户可以在家庭实验室中进行结晶程序，并将合适的结晶板带到光束线上。对于某些样品，确定合适的数据收集参数可能很重要，特别是在辐射损伤是一个问题的情况下。在大多数情况下，自动数据处理完全足以回答科学问题，尽管用户保留了使用光束线工具进行重新处理的能力，例如，空间群不明确或仅使用收集数据的初始部分来最大限度地减少辐射损伤影响。
如果初始结晶试验中没有产生合适的晶体，则可以探索蛋白质浓度、纯度或结晶筛选的变化，也可以使用晶体晶种。如果晶体在光束线上没有衍射到有用的分辨率，则可以将网格扫描与未衰减的光束一起使用，以评估晶体的固有衍射极限和晶胞，以指导优化工作。太小而无法在板内收集数据的晶体（例如，<10 μm）可能适用于连续晶体学或纳米焦点实验（例如，在金刚石光束线VMXm处）。使用 VMXi 数据求解结构通常通过分子替换变得简单明了，特别是自从 Alphafold16 出现以提供有效的搜索模型以来。如果这不成功，可以从板中收集晶体并冷冻冷却，以实现常规的单波长异常衍射、多波长异常衍射或长波长相位实验。
这种方法的优点包括能够直接从结晶板获得快速、高质量的数据集和反馈，而无需干扰晶体生长的环境。结构生物学中所谓的"室温复兴"重视在非低温条件下获得的结构，以便探索更多的生理相关性和蛋白质动力学2.通常，与优化的低温冷却晶体相比，其分辨率略低，但前提是已经建立了合适的冷冻条件，并且晶体对机械处理和结晶液滴3 的打开具有鲁棒性。该管线非常适合的即将到来的应用是在药物发现中在室温下大规模筛选蛋白质-配体复合物或片段活动。配体或片段可以共结晶，也可以在室温数据收集之前通过移液器或声滴喷射添加。另一个应用是以高效的方式快速测量来自数百或数千个晶体的数据，然后使用 DIALS17 multiplex14 软件提取可能代表不同生物实体的同晶簇，或建立以不同方式处理或暴露于不同配体或信号的晶体群体之间的统计学显着差异。

X射线晶体学仍然是了解蛋白质结构和功能的关键工具，可提供蛋白质或其复合物的高分辨率结构，例如底物或候选药物。然而，在许多情况下，获得具有理想特性的晶体 - 高度衍射，适合浸泡的晶体形式，并且没有晶体病理，如孪晶 - 仍然是一个相当大的瓶颈1。由于通常无法预测生产蛋白质晶体的合适化学条件，因此探索数千种潜在化学混合物的结晶筛选是标准的，通常由自动化/机器人辅助设置屏幕和晶体酒店，以监测，通常是远程，记录的结晶液滴图像。
当晶体出现时，通常必须使用尼龙或卡普顿环从结晶环境中收集晶体，然后转移到含有冷冻保护剂的液滴中（搜索是一个额外的变量），然后再冷冻到液氮中。结晶和 X 射线数据收集之间的这些额外步骤可能涉及当其密封环境破裂时结晶液滴的脱水、处理晶体时晶体上的机械应力以及冷冻保护剂对晶格的损坏（通常导致镶嵌扩散增加）等因素2.此外，晶体采集是时间和劳动密集型的，并可能导致样品之间的不均匀性，尤其是在采集过程中液滴上形成皮肤时。VMXi 光束线可以从粘附在板上的晶体访问可用数据，否则这些数据将被丢弃以进行数据收集。
绝大多数 X 射线晶体结构都是使用上述方法在 100K 下确定的，从而实现了直接的晶体传输和处理，并将 X 射线束中的晶体寿命提高了几个数量级。然而，人们越来越关注在非低温条件下确定结构，即更接近与蛋白质功能相关的生理条件2,3,4。这样可以更好地理解蛋白质的动态结构，避免氨基酸构象或环在功能不相关的状态下被冻结5，并使配体结合能够在更接近细胞和生物体内蛋白质自然环境中的条件下进行探索6。
在英国金刚石光源同步加速器的多功能大分子晶体原位 （VMXi） 光束线上实施的另一种方法是，在环境条件下，在没有干扰的情况下，直接从晶体生长的环境（即结晶板内）测量晶体的衍射数据 7,8.这样可以非常快速地从结晶筛选和优化中获得反馈，以指导用户获得满足其要求的最佳晶体形式。它还能够以自动化方式生产高质量的室温结构9.
该协议假设用户具有准备结晶的高纯度蛋白质样品。我们描述了访问Harwell结晶设施以生产蛋白质晶体，然后使用光束线VMXi进行数据收集的用户体验（图1）。
哈韦尔的结晶设施
Harwell （CF） 的结晶设施位于 Harwell 研究综合体 （RCaH），毗邻金刚石光源。该设施为用户提供了一个用于大分子结晶的高通量自动化实验室，使用机器人技术进行结晶筛选、晶体优化、晶体成像和表征。通过与高度自动化的VMXi光束线紧密集成，确定室温结构的速度大大加快，并能够在非低温条件下表征新型蛋白质结构、蛋白质-配体和DNA-配体复合物，以及自动片段筛选（图1）。
CF管道是一套仪器，包括用于可溶性和膜蛋白结晶的纳升结晶机器人9 ，用于制备商业结晶筛和复杂定制优化筛的液体处理机器人，以及四台成像仪器（一台在4°C，三台在20°C下用于结晶板的成像（参见 材料表).一台成像仪能够对脂质立方相 （LCP） 玻璃板进行成像，一台成像仪配备多荧光光学元件（均在 20 °C 下）。
该设施现在被广泛的学术和工业用户广泛使用，包括膜蛋白实验室（MPL;https://www.diamond.ac.uk/Instruments/Mx/MPL.html）、XChem 片段筛选设施 10、MX 光束线、XFEL 集线器以及罗莎琳德富兰克林研究所 （RFI）。这种成熟和优化的管道使结晶实验能够在广泛的结构生物学项目中进行。本文描述了用于 VMXi 数据收集的晶体管道，尽管晶体也可以被收集和冷冻冷却或定向到 XChem 管道。
用户访问通过钻石MX提案系统（https://www.diamond.ac.uk/Instruments/Mx/Synchrotron-Access.html）分配，工业用户通过钻石行业联络小组获得支持。所有用户都可以带着他们的样品或板来到现场，这些样品或板可以手工运输。不建议通过快递发送板，因为我们的经验表明，液滴可能会从分配位置移开，或者液滴可能会被结晶储液器损坏。或者，通过安排，用户可以将他们的蛋白质样品送到CF，在那里工作人员代表他们进行结晶实验。对于CF而言，用户可以通过登录Rock Maker Web或通过ISPyB（对于VMXi）来远程监控实验。CF的访问可以基于Diamond收集的X射线衍射结果以迭代方式进行。
金刚石光源的光束线VMXi
光束线VMXi（以下简称"光束线"）是一种独特的、最近开发的仪器，完全致力于室温、高度自动化的X射线晶体学，重点是测量合适结晶板内晶体的数据。光束线提供微焦点 （10 x 10 μm）、粉红色光束（带通 <5 × 10-2ΔE/E），高通量为 ~2 × 1013 个光子/秒（16 KeV）7。这种高通量光束与快速检测器相结合，可实现非常高的样品通量，并可从尺寸超过 10 μm 的样品中收集数据。
结晶板通过存储在样品存储系统中进入光束线，并根据用户提供的时间表进行成像，同时使用 ISPyB11 接口 SynchWeb12 对板进行配准。通常，建议用户选择斐波那契数列的时间点进行成像（0、12、24、36、60...从进入系统的板起 7,320 小时）。一旦对板进行成像，用户就会收到电子邮件通知。可见光和紫外光成像均可按需提供给用户。通过机器学习算法分析样品存储系统拍摄的图像;这会自动定位和定义类似于晶体的对象的兴趣点，并注册兴趣点，以便用户将其添加到队列中以进行数据收集。用户也可以手动单击可见光图像以注册兴趣点，也可以单击并拖动要通过光栅扫描进行分析的区域。用户可以将这些点与自动定位的点一起添加到队列中。
一旦所有样品都具有用于数据收集的适当参数，板就会进入队列。当板到达队列的顶部时，它会自动分配到光束线。结晶板由机械臂自动从晶体酒店加载到光束线中，在图像匹配之后，根据用户定义的说明从每个选定的晶体测量高达60°旋转的晶体学数据集。板内的所有液滴都可用于光束线上的这些实验。数据从多个晶体合并，以自动方式生成同构的、最佳合并的数据集 7,9。收集所有排队的数据集后，用户将收到一封电子邮件，其中包含一个链接，可以查看 ISPyB11 中的数据集，就像在其他 Diamond MX 光束线中一样。用户也会被定向到光束线网页 （https://www.diamond.ac.uk/Instruments/Mx/VMXi.html）。

<table><tbody><tr><td>Formulator</td><td>Formulatrix</td><td>on request</td><td>Liquid handling robot</td></tr><tr><td>Formulatrix imager</td><td>Formulatrix</td><td>on request</td><td>Crystallisation plate imager</td></tr><tr><td>Greiner CrystalQuick X&amp;nbsp;</td><td>Greiner</td><td>Z617644</td><td>Crystallisation plate</td></tr><tr><td>Gryphon&amp;nbsp;</td><td>Art Robbins Instruments</td><td>620-1000-10&amp;nbsp;</td><td>Crystalisation robot</td></tr><tr><td>MiTeGen Insitu-1</td><td>Mitegen</td><td>InSitu-01CL-40</td><td>Crystallisation plate</td></tr><tr><td>Mosquito LCP&amp;nbsp;</td><td>(SPT Labtech)</td><td>on request</td><td>Crystallisation robot</td></tr><tr><td>Rock Imager &amp;amp; Maker</td><td>Formualtrix</td><td>on request</td><td> Software for Imager&lt;br/&gt; [1] https://formulatrix.com/protein-crystallization-systems/rock-maker-crystallization-software/ </td></tr><tr><td>Scorpion</td><td>Art Robbins Instruments</td><td>640-1000-10&amp;nbsp;</td><td> Liquid handling robot&lt;br/&gt; https://www.artrobbins.com/scorpion </td></tr></tbody></table>

crystallization and in situ room temperature data collection using the crystallization facility at harwell and beamline vmxi, diamond light source

描述了使用Harwell的结晶设施进行机器人蛋白质结晶的方案，以及从金刚石光源光束线VMXi的结晶板 中原位 室温数据收集的方案。这种方法能够以直接的方式从多个晶体中确定高质量的室温晶体结构，并对结晶试验的结果提供非常快速的反馈，并实现连续晶体学。室温结构在理解蛋白质结构、配体结合和动力学方面的价值在结构生物学界越来越得到认可。来自世界各地的用户可以通过多种可用的访问模式访问此管道。设置的结晶实验可以进行成像和远程查看，并使用机器学习工具自动识别晶体。数据在基于队列的系统中进行测量，该系统具有来自孔板中用户选择的晶体的多达 60° 旋转数据集。来自特定孔或样品组中所有晶体的数据使用 xia2.multiplex 自动合并，输出可通过 Web 浏览器界面直接访问。

结晶设施和 VMXi 光束线已用于各种项目类型和用例。以下是一些示例来说明用户可能希望追求的内容。
案例研究 1: 标准数据收集
光束线能够从结晶板内的少量晶体中快速确定室温晶体结构。晶体的最小数量取决于空间群和晶体取向，但通常为1-4个，尽管可以通过合并来自几十个晶体的数据来提高数据质量。最近的一个例子是光束线标准之一，thaumatin。如 图8A所示，多个晶体被标记为手动收集数据，如协议第2.3节所述。如协议第2.4节所述，将这些晶体添加到队列中，并从下拉列表中选择实验参数。一旦应用了实验参数，板就会排队等待数据收集。数据集使用 xia2.multiplex 管道收集、自动缩放和合并，如协议第 3 节所述。SynchWeb 的输出示例如 图 8A 中间所示。五个合并的数据集产生了分辨率为 1.66 Å 的数据集。对于孔中大约五个晶体的标准数据收集，数据集在 2.5 分钟内收集。
案例研究 2:配体结合 – 使用 Mac1 蛋白的片段实验
使用光束线可以直接在室温下产生蛋白质-配体复合物的结构。配体可以添加到结晶板上的液滴中（手动或通过声滴注入），并在适当的孵育时间后测量数据。在此描述的示例中，将一系列片段分配到含有结晶板中蛋白质 nsp3 （Mac-1） 的 SARS-CoV-2 第一大结构域晶体的孔中。如方案步骤2.5所述，将含有相同片段的两个孔分配为一组。如方案步骤2.3和2.4所述，标记多个晶体（42）以进行数据收集，并使用标准参数（60°旋转，0.1°步长，0.00178秒曝光，5%透射率，每个晶体16 KeV）收集数据集（图8B）。来自两个井的数据集使用 xia2.dials 管道自动处理，随后，启动 xia2.multiplex 管道以自动合并其中的 22 个数据集。然后，在这些管道的输出上运行DIMPLE，并生成地图，清楚地显示了绑定片段的证据。碎片模型被内置到未占用的密度中并进一步细化（图8B 右）。使用这一系列步骤可以很容易地确定室温配体结合结构，为基于结构的药物设计过程提供宝贵的信息和反馈。对于跨多个孔的 42 个晶体的数据收集，数据集在 10 分钟内收集。
案例研究 3:具有低对称空间群和优选取向的结构解 使用c型气体结合细胞色素进行结晶实验，产生具有板状形态的多个晶体堆叠（图8C）。通过在X射线束中只有一个晶体的堆栈边缘选择几个位置，通过合并来自四个晶体的楔块，可以获得分辨率为1.75 Å的高质量数据集，尽管存在单斜晶系（C2）空间群。这使得项目能够快速推进，而无需进一步优化结晶条件。这个结果在前面已经描述过9.对于孔中四个晶体的数据收集，在 2 分钟内收集了数据集。
案例研究 4:使用连续晶体学从板中的微晶中获取信息和室温结构
通常，当微晶以液滴形式出现时，或者当用户寻求批量优化微结晶方案时，作为同步加速器或XFEL源连续晶体学实验的前体，使用最少的材料获得有关不同试验的衍射特性和晶胞尺寸的快速反馈非常有帮助。在此用例中，将批量生长的溶菌酶微晶移液到结晶板（每滴200 nL体积）中，并使用步长为10μm的网格扫描从8液滴中收集数据（图9）。使用串行晶体学软件处理得到的 25,906 张静止图像，从而形成一个数据集，其中 9,891 个衍射图被索引并合并，生成一个分辨率为 2.0 Å 的数据集，该数据集根据已发表的室温结构进行了很好的改进（使用 PDB 8A9D 时，R功 = 19.6%，无 R = 23.6%）（表 1).这允许对晶胞分布进行详细分析和微晶室温结构测定，从而可以用于复杂的连续晶体学实验，包括时间分辨研究。所需微晶悬浮液的总体积为 1.6 μL。对于使用网格扫描在八个孔中收集微晶的数据，在 40 分钟内收集了数据集。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65964/65964fig1.jpg"> 图 1:蛋白质到结构管道的示意图，该管道集成了结晶筛选、结晶设施的优化、VMXi 的室温自动数据收集和处理（无需样品采集）、XChem 片段筛选以及其他 MX 光束线的数据收集。 用户可以通过提供样品或将板带到 VMXi 光束线来启动管道。缩写:Versatile Macromolecular Crystallography in situ。 <a href="https://www.jove.com/files/ftp_upload/65964/65964fig1large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65964/65964fig2.jpg"> 图 2:用于设置结晶板的 Mosquito SPT Labtech 界面。 （一） （1） MiTeGen 原位 1 设置视图。通过转到 （2） 96 孔板类型并选择 （3） MiTeGen 板 2 滴板来选择 MiTeGen 2 滴标准板。要更改 VMXi 所需的删除 1 和删除 2 的定义参数，请单击 （4） 编辑图标。这将打开一个新窗口 （B），其中 （5） X 和 Y 偏移需要更改，如图所示。选择 （B） 子孔 2 和 （C） 子孔 3 并相应地更改值。（D） CrystalQuickX 设置视图。通过转到 96 孔板类型并选择 MiTeGen 板 2 滴板来选择 CrystalQuickX 2 滴标准板。要更改 VMXi 所需的放置 1 和放置 2 的定义参数，请单击与上述相同的编辑图标。这将打开一个新窗口，其中 （E，F） X 和 Y 偏移需要更改，如图所示。<a href="https://www.jove.com/files/ftp_upload/65964/65964fig2v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65964/65964fig3.jpg"> 图 3:SynchWeb 界面显示了如何创建 VMXi 货件、注册铭牌和查看联系方式。 （A）下拉菜单，（B，C）注册新货件，（D）注册新集装箱，（E）输入车牌信息，（F）检查联系方式，以及（G）提案中已注册的集装箱列表，显示了将信息上传到SynchWeb界面的各个阶段的屏幕截图。 <a href="https://www.jove.com/files/ftp_upload/65964/65964fig3large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65964/65964fig4.jpg"> 图 4:使用 SynchWeb 选择和准备用于数据收集的样本。 将显示一系列屏幕截图，其中显示了使用 SynchWeb 界面准备用于数据收集的样品的各个阶段。（A） 从液滴概览中选择感兴趣的点和区域。在这个面板的下半部分，有一个按时间顺序排列的一滴照片系列。（B） 一块板的 CHiMP 输出示例，突出显示了"晶体"类别的结果。（C） 从选定点和区域列表中将样本添加到队列中，以及 （D） 从光束线创建的实验设置的下拉列表中将数据收集参数应用于排队的样本。注意没有实验参数的样品（红色）与正确应用参数的样品（顶部和底部）之间的差异。该面板的底部是 "排队容器 "按钮，用于将要在光束线上收集的板排队。 <a href="https://www.jove.com/files/ftp_upload/65964/65964fig4large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65964/65964fig5.jpg"> 图 5:在 SynchWeb 中创建示例组。 一系列屏幕截图，显示了创建示例组的各个阶段。（A） 从相关货物中选择含有样品的板，以及 （B） 选择板内的液滴。这些可以是单独的水滴，也可以按行和/或列选择。（C） 已创建的样本组列表。（D） 列出最后三个多路复用处理作业的输出，并可以选择这些输出以显示处理管道中的统计信息。 <a href="https://www.jove.com/files/ftp_upload/65964/65964fig5large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65964/65964fig6.jpg"> 图 6:数据处理和数据缩减。 （A） ISPyB11 中已处理数据集的屏幕截图。用于访问再处理功能的按钮将突出显示。样品ID和实验参数显示在左上角，衍射图像查看器显示在中间。单击此图像将打开一个交互式窗口来检查不同的图像。晶体图像查看器显示在右侧，单击此图像还将打开一个交互式窗口，用于比较光束线和 Formulatrix 存储图像。每张图像的分析图显示在最右侧，单击此图像将打开此输出的放大版本。单击" 自动处理 "选项卡将使自动处理可见，并使不同管道的结果之间的比较变得容易。单击选项卡可在不同的处理管道之间切换，并查看所选管道的详细输出。用于数据下载的 "日志和文件 "按钮将突出显示。单击" 下游处理" 选项卡将展开，并在适当的情况下为通过数据缩减后管道运行的任何数据集提供结果。（B） 样品组管理 屏幕的屏幕截图。用户定义的组名称位于顶部，所包含井的视觉描述如下。绿色孔表示从该液滴中测量的所有晶体都将包含在该组中。可以看到对该组执行的不同多路复用作业的摘要，下面是多路复用的详细输出。" 数据集 "按钮将突出显示，用于检查包含的实验。 <a href="https://www.jove.com/files/ftp_upload/65964/65964fig6large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65964/65964fig7.jpg"> 图 7:数据再处理窗口。 （A） 单个和 （B） 多晶数据集。将显示两个单独的数据集，其中选择了数据区域。选中" 单独处理 "复选框后，将按 "集成 "按钮单独处理所选的衍射图像。单击 "多晶" 按钮将打开各个数据集的显示。要重新处理来自多个数据集的衍射图像，需要选择显示的图像区域，然后单击突出显示的 "集成" 按钮来启动重新处理。 <a href="https://www.jove.com/files/ftp_upload/65964/65964fig7large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65964/65964fig8.jpg"> 图 8:VMXi 管道的代表性结果。 （A） 结晶液滴中蛋白质索马汀的标记晶体（左图）、数据处理结果（中图）和电子密度（右图）。（B） 收集多个晶体以确定片段与 SARS-CoV-2 宏结构域的结合。使用标准实验设置，在存在来自EU-OPENSCREEN片段屏幕的片段的情况下，在多个晶体上收集数据集。这些数据集合的示例显示在 SynchWeb 的摘录中。碎片被构建成相应的密度，并进一步细化，如右侧最远处所示。（C） 用于数据收集的具有挑战性的结晶命中的堆栈中标记的单斜晶体。绿色十字和红色数字表示使用 10 μm 光束和 60° 旋转测量数据的位置。将得到的四个楔块合并，以生成分辨率为 1.75 Å 的数据集。血红素基团周围的电子密度显示在右侧。 <a href="https://www.jove.com/files/ftp_upload/65964/65964fig8large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65964/65964fig9.jpg"> 图 9:结晶板中的连续晶体学。 （A） 结晶液滴的光学图像，白色框代表感兴趣的区域。（B） 网格扫描点的定义。（C） 表示衍射的热图。（D） 由超过 9,000 个静止衍射图谱的连续晶体学数据集产生的电子密度图。 <a href="https://www.jove.com/files/ftp_upload/65964/65964fig9large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>分辨率 （Å）</td>
			<td>完整性 （%）</td>
			<td>多样性</td>
			<td>I/σ（I）</td>
			<td>R拆分 </td>
			<td>CC1/2 （英语） </td>
			<td>独特的观察结果</td>
		</tr><tr><td>整体</td>
			<td>100</td>
			<td>95.5</td>
			<td>20.8</td>
			<td>0.063</td>
			<td>0.998</td>
			<td>8422</td>
		</tr><tr><td>最低价 （55.55 - 5.43）</td>
			<td>100</td>
			<td>147.1</td>
			<td>81.7</td>
			<td>0.028</td>
			<td>0.999</td>
			<td>488</td>
		</tr><tr><td>最高价 （2.03 -2.00）</td>
			<td>100</td>
			<td>75.3</td>
			<td>1.2</td>
			<td>1.092</td>
			<td>0.410</td>
			<td>411</td>
		</tr></tbody></table>表 1:VMXi RT 串行数据集的数据统计信息。缩写: I = 比例观测值的平均强度;R分裂 = 测量强度差异的量度;CC 1/2 = 数据集的两个随机半部分之间的相关系数。

Watch this Scientific Journal Video about Advancing Protein Structure Analysis for Drug Development at JoVE.com

作者聚焦:推进蛋白质结构分析用于药物开发 

我们提出了一种使用哈韦尔研究综合体中的结晶设施进行蛋白质结晶的方案，以及随后在 Diamond 的多功能大分子晶体原位 （VMXi） 光束线中从板内晶体中收集的原位 X 射线晶体学数据。我们描述了样品要求、结晶方案和数据收集指南。

使用 Harwell 的结晶设施和 Beamline VMXi（金刚石光源）进行结晶和 原位 室温数据收集

Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from ...

了解蛋白质的三维结构是药物设计、生物技术以及我们对许多不同疾病的理解的基础。VMXI 无需冷冻冷却即可确定蛋白质结构，因此更接近其自然温度。VMXI 还可以在蛋白质晶体的结晶板中测量其衍射数据，从而避免机械处理造成的任何损坏。在未来，我们真的希望利用VMXI令人难以置信的吞吐量来探索大量的晶体，以识别许多不同的结构状态，而不是人们现在经常获得的单一结构。我们还希望推动药物开发，并在结晶板内进行片段筛选和优化。一种显示出巨大前景的新方法。

crystallization-situ-room-temperature-data-collection-using

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Crystallization and In Situ Room Temperature Data Collection Using the Crystallization Facility at Harwell and Beamline VMXi, Diamond Light Source

3.4K 次观看.  Harwell Science and Innovation Campus. 了解蛋白质的三维结构是药物设计、生物技术以及我们对许多不同疾病的理解的基础。VMXI 无需冷冻冷却即可确定蛋白质结构，因此更接近其自然温度。VMXI 还可以在蛋白质晶体的结晶板中测量其衍射数据，从而避免机械处理造成的任何损坏。在未来，我们真的希望利用VMXI令人难以置信的吞吐量来探索大量的晶体，以识别许多不同的结构状态，而不是人们现在经常获得?...

视频: 作者聚焦:推进蛋白质结构分析用于药物开发 

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.

Automated systems and protocols for the routine preparation of a large number of screens and nanoliter crystallization droplets for vapor diffusion experiments are described and discussed.

分子生物学实验室中大分子结晶的自动化协议

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

生物大分子晶体的完全自主表征及数据采集

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Fixed Target Serial Data Collection at Diamond Light Source

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is presented with detailed step-by-step instructions, figures, and videos for smooth data collection.

We present a comprehensive guide to fixed target sample preparation, data collection, and data processing for serial synchrotron crystallography at Diamond beamline I24.

金刚石光源的固定目标串行数据收集

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise.
Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. 
We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

VMXm 光束线微晶样品制备管道

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 &#197;. 
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. 
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

金刚石光源下光束线I23真空长波长晶体学的样品制备和转移实验方案

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

通过微透析对芯片上的蛋白质进行结晶，用于 原位 X射线衍射研究

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

化学

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

室温下的片上结晶和大规模连续衍射

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. 
It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

This protocol demonstrates how to image biological cryo-preserved samples using cryo-structured illumination microscopy. We demonstrate the methodology by imaging the cytoskeleton of U2OS cells.

低温结构照明微观数据收集来自低温保存细胞

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than ...

使用 Harwell 的结晶设施和 Beamline VMXi（金刚石光源）进行结晶和原位室温数据收集

副本

摘要

探索更多视频

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到

分子生物学实验室中大分子结晶的自动化协议

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

生物大分子晶体的完全自主表征及数据采集

金刚石光源的固定目标串行数据收集

VMXm 光束线微晶样品制备管道

金刚石光源下光束线I23真空长波长晶体学的样品制备和转移实验方案

通过微透析对芯片上的蛋白质进行结晶，用于原位 X射线衍射研究

室温下的片上结晶和大规模连续衍射

低温结构照明微观数据收集来自低温保存细胞

使用 Harwell 的结晶设施和 Beamline VMXi（金刚石光源）进行结晶和 原位 室温数据收集

副本

摘要

探索更多视频

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到

分子生物学实验室中大分子结晶的自动化协议

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

生物大分子晶体的完全自主表征及数据采集

金刚石光源的固定目标串行数据收集

VMXm 光束线微晶样品制备管道

金刚石光源下光束线I23真空长波长晶体学的样品制备和转移实验方案

通过微透析对芯片上的蛋白质进行结晶，用于 原位 X射线衍射研究

室温下的片上结晶和大规模连续衍射

低温结构照明微观数据收集来自低温保存细胞

使用 Harwell 的结晶设施和 Beamline VMXi（金刚石光源）进行结晶和原位室温数据收集

通过微透析对芯片上的蛋白质进行结晶，用于原位 X射线衍射研究