有很多谁促成了这一工作，大部分是从膜的结构和功能的生物集团，无论是过去和现在的成员。我们致以热烈的感谢和赞赏。这项工作是支持的，部分资金从科学基金会爱尔兰（07/IN.1/B1836人），美国国立卫生研究院（GM75915，P50GM073210的和U54GM094599），FP7 COST行动CM0902。

1。准备的结晶板设置机器人做结晶试验开始与制备的基板的玻璃夹心结晶板（ 图2），在参考文献2中详细描述。底板必须首先被硅烷化双棍的多孔隔板，创建的井，必须施加到板。根据材料逐项列出需要的材料和用品。 <ol><li>将板在纸巾上，适用几滴硅烷化溶液，均匀的板面用薄纸分发。 </li><li>板浸在烧杯中的水，去除多余的硅烷化解决方案。 </li><li>板的烧杯，将其放置在干燥的纸巾，硅烷化的一面，而其暴露的表面干燥用纸巾轻轻擦拭。 </li><li>剥离保护盖的穿孔，双棍间隔和应用的隔离物，粘性侧下降，干燥，硅烷化的玻璃板表面。成为确保间隔件的底板上正确的位置，与上部左手角落和两个相邻的两边的间隔件和板，重合。 </li><li>使用的辊或BRAYER平坦的间隔件的底板上，以建立它们之间的紧密密封。 </li></ol>预组装板市售（HR3-151，汉普顿研究MD11-55-100;，分子尺寸和LCP96T-UVP70U Swissci）。出于成本的原因，我们更愿意把我们自己的组装板后，上述协议。 （2） 在细观机器人1 观机器人被证明在这个视频文章中的第一个，是一个目前正在使用的MS＆FB组（ 图3）。细节的机器人，可根据材料和参考第5。的材料和成套设备NT需要使用的机器人是根据材料逐项建立的结晶试验。 <ol><li>将机器人的甲板上的第1节中制备与间隔件的底板。它有自己的平台，标志着底板对齐的目的，具有良好的位置。板应标记，以资识别。 </li><li>固定96孔沉淀剂的块在其自己的平台的甲板上的机器人旁边的结晶板。 </li><li>小心打开块脱落的塑料密封帽。 </li><li>打开加湿器和指示在甲板上的机器人的底板和跨潮湿的空气流的。已被证明的加湿以减少沉淀剂溶液的蒸发，并增强跨越的结晶板5的重现性。它可以被打开之前设置预加湿的区域环境相对湿度低时，或以其他方式，就在中间相开始分配。 </li><li>初始化机器人按照制造商的说明。仪器的具体说明不会在任何细节，因为确切的程序会随时间而改变。从本质上讲，期间发生初始化的是，在机器人上的分装臂的中间相，在三个正交方向X，Y和Z设置有基准位置</li><li>将蛋白质的载货中间相微量注射器，点胶针，手臂的机器人（ 图3）。如何准备的中间相，以及如何将其加载到注射器中的全部细节已在参考文献3,7-10。 </li><li>从隔板上的结晶底板拆下上部的保护盖，和在平板上的孔对齐，基准点上的底板平台。注意，对于该视频的目的，保护盖留在原地，以帮助在查看器的取向步骤期间。 </li><li>对齐分配希瑞恩的针尖e的底部以及在左手角落的结晶板（孔A1）的最上面。这是通过眼调节的高度或Z-坐标的分装臂一边看如何接近的针尖来的阱的底部。可以判断的平台上，而有一点点调节分配的手臂的高度，从一侧向另一侧移动板的尖端刚刚接触到井底。 </li><li>输入到计算机中的分装臂的前端接触的板的Z-坐标。配药被编程，以在从该基准的Z位置对应的基板的表面的80微米与针尖。 </li><li>由眼睛的针尖在中心孔A1的位置。储存在控制计算机的参考X，Y坐标的分配臂尖为中心。此坐标被用来定义盘上剩余的井的中心。 </li><li>镨输入法使用适当的命令机器人计算机上，以确保新鲜的中间相填充的注射器的分配针和第一和随后的井接收其全部的中间相。挤压中间相的针尖，用纸巾擦去。 </li><li>启动运行机器人（X-AP）计划发起了一轮的沉淀分配的机器人臂的提示进行清洗，并准备吸解决方案的沉淀块的冲洗步骤。 </li><li>激活分配函数在机器人上设置在运动顺序装载与载货蛋白的中间相（通常是50 nl）的第一，八口井的列，在一个时间，然后通过沉淀剂溶液（通常为800 nl）的水井。加载列之间，中间相送出臂返回到停车位置的针头末端被放置在一个湿的海绵干燥，以防止中间相的尖端处。配药中间相和沉淀剂的方法，重复横跨板11次，直到板填充。与中间相和沉淀剂的装载的96孔板中以5分钟来完成。 </li><li>只要填充板和机器人手臂已经回到公园的位置，取出盘子从甲板上的机器人，并把它放在一个平坦的固体表面。 </li><li>随即，密封板的盖玻璃，以及组合物的随时间的变化最小化。重要的是要确保完全覆盖所有96个孔，该玻璃盖对准方形与底板和盖玻璃，没有延伸超出底板的足迹。甲的辊或BRAYER用于建立紧密和均匀的密封件之间的盖玻璃和隔板。 </li><li>检查板，可以肯定的，因为它应该去设置。什么人找的是一个“炸蛋上的泛在每口井的外观。炒鸡蛋的蛋黄相对应的中间相。白色的蛋是沉淀的解决方案和平底锅的边缘的周长是结晶良好。在理想的情况下，三是同心的。 </li><li>板放置在用于晶体生长的温度 - 控制的存储区域。 </li><li>如果没有更多的板块设置，删除的沉淀块从甲板的机器人，封紧袋口，并把它放回存储。 </li><li>包含中间相从分配的机器人手臂上取下注射器，拆除，部分，包括针头和套圈，仔细洗，甲醇和干，准备以备下次使用。 </li></ol> 3。 在细观机器人2中。蚊子LCP 蚊子LCP的机器人（ 图4）采用类似的中间相分配器机器人1所使用的。相反的机器人，它通过分发沉淀的一次性吸头。使用编程软件来设置仪器工作与特定的的结晶板和precipit蚂蚁块，在一个指定的序列，以及免除用户定义的卷的中间相和沉淀剂。这样的细节由生产商所提供的，并在这里将不进行描述。 <ol><li>准备一个Hamilton注射器，完全用针和充满蛋白质载货中间相，如上述那样根据机器人1和JOVE文章1712 1。 </li><li>夹紧位置上的分装臂的机器人的注射器。 </li><li>使用安装在甲板激光传感器建立分配注射器的针上的X和Y坐标。这是一个重要的步骤，以确保准确地被分配的中间相上的板在各孔的中心。 </li><li>随着触摸控制面板上的一个按钮，总理的注射器的针排出中间相。取出挤压的中间相从针尖与组织中准备用于使用机器人井加载。 </li><li>前放置在甲板上的机器人的湿润的结晶板连接平台，用几滴的水，以帮助保持板在毛细作用的表面。 </li><li>从间隔的玻璃底板上取下保护盖，并将其放置在其平台上牢固，确保良好的附着力。在平台上使用的三个基准点对准3角井，从而确保适当的定位孔的上盘相对于分送针尖。该仪器现在命令输入分配周期。 </li><li>第二个吸步骤中间相被分配到一个小玻璃盘前加载井用新鲜的中间相周期的开始。的机器人第一中间相负载时的盘上的8孔一列垂直的，然后使用一次性吸头的顶部分配沉淀剂溶液的中间相丸剂。沿板的长度，重复该过程11次，直到所有的井填充。整个过程大约需要5分钟才能完成。 </li><li>清除从机器人平台填充板和密封用的盖玻璃，如上所述，根据机器人1。 </li><li>板放置，适当标记的，在晶体生长的温度控制环境。 </li></ol> 4。 在细观机器人3。狮鹫LCP 狮鹫LCP的机器人（ 图5）采用上面所描述的机器人1和2类似的中间相分配系统。什么区别于其他两个是同时，所有96个沉淀的解决方案中分配。 <ol><li>准备载货的蛋白质-中间相，将其加载到分注注射器中，如上所述，根据机器人1和2，并在JOVE文章1712 1。 </li><li>夹紧位置上的分装臂的机器人的注射器。 </li><li>沉淀剂溶液块和结晶板放置在甲板上的机器人。 </li><li>装入96尖HEAD与沉淀的解决方案。用户定义的延迟时间，以方便吸粘性沉淀。 </li><li>命令仪器进入点胶周期。周期开始时，中间相顺序被分配到一个过程，需要大约1分钟完成的96口井在。然后，该96尖头一次分配所有96沉淀剂的解决方案的顶部的中间相在结晶井的丸药。整个板块在加载过程结束后2分钟。 </li><li>从机器人平台卸下填充板和密封用的盖玻璃，如上所述，根据机器人1。 </li><li>板放置，适当标记的，在晶体生长的温度控制环境。 </li></ol> 5。代表性的成果此视频文章的目的是演示如何设置自动结晶实验，带有粘性的，膜蛋白载货的中间相的玻璃sandwic小时96孔结晶板。强调的是自动化，通过使用专门的机器人，需要最少的人工干预。可重复性和精确性，大量的中间相和沉淀分配，而在井的位置，当机器人。这些都是重要的从点有效的筛选条件，支持crystallogenesis视图。他们也分析管道中的下一步骤是评估经常做的晶体生长与成像设备的关键。下降或静脉推注的位置是关键的成像过程，，不规则放置不当的丸井通常需要用户干预以纠正，并需要一定的时间。的正确和不正确的加载板的实施例用于说明的目的， 如图6中所示。 <img src="/files/ftp_upload/4000/4000fig1.jpg" alt="图1" /> <html> 图1的流程图总结了在生产，收获和低温冷观种植的膜蛋白晶体所涉及的步骤。红色虚线包围的步骤是此朱庇特的文章关注的焦点。机器人用，步骤29-32，免除中间相和沉淀的解决方案。从参考文献3。 <a href="http://jove.com/files/ftp_upload/4000/4000fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/4000/4000fig2.jpg" alt="图2" /> 图2，一种完全加载并密封的96孔玻璃夹心结晶板。在各孔中含有50 NL立方相和1μl的沉淀剂溶液。为了清楚起见，立方相已与苏丹红染色和沉淀剂溶液包括亚甲基蓝。从参考文献5。 _upload/4000/4000fig3.jpg“ALT =”图3“/&gt; 图3： 在，细观机器人（机器人）。该机器人有两个武器。臂1分注蛋白质充满油脂的中间相。臂分配沉淀的解决方案。在填充过程中，在96孔板中保持固定在甲板上的机器人本身是固定的位置。两只手臂移动，提供中间相和沉淀的解决方案。从参考文献3。 <img src="/files/ftp_upload/4000/4000fig4.jpg" alt="图4" /> 图4。蚊子：LCP机器人（机器人2）。在机器人1，蚊子的LCP的甲板，赖以坐的96孔板中和沉淀剂溶液块相比，在填充过程中水平移动。 <img src="/files/ftp_upload/4000/4000fig5.jpg" alt="图5" /> 图5。：狮鹫，LCP机器人（机器人3）。在一个成立，脂质立方相分配器转移到96孔板中，以提供蛋白质的载货的中间相。甲板上，​​然后翻译水平的沉淀剂溶液尖头定位下盘。同时配发了96沉淀的解决方案。的图片来自制造商（ <a href="http://www.artrobbins.com/" target="_blank">http://www.artrobbins.com/</a> ）和标记。 <img src="/files/ftp_upload/4000/4000fig6.jpg" alt="图6" /> 图6。完全装载和密封的96孔玻璃夹层板（A）和井正常（B）和不正确（C，D）使用在，细观机器人（机器人）的填充。板在条形码标签上A用于跟踪板在板孵化器/成像设备。面板的BD中，沉淀剂溶液和中间相的标记P和M分别。在锅上的预期的&#39;炒鸡蛋以及在B的外观是一个适当的加载和密封良好的特征。在C中，沉淀剂，已被分配入井不当在偏离中心的位置，并在与隔离物接触来。在C语言中，它会显得过于中间相的体积比它应该是。 D.对于规模与沉淀剂溶液的不完全加载的一个例子中所示，所有的孔的直径为6毫米。

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USA. 107, 1077-1081 (2010).</li><li>Cherezov, V., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picolitre-scale+crystallization+of+membrane+proteins.">Picolitre-scale crystallization of membrane proteins.</a> J. Appl. Crystallogr. 39, 604-606 (2006).</li><li>Li, D., Boland, C., Aragao, D., Walsh, K., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+cryo-cooling+crystals+of+membrane+proteins+grown+in+lipidic+mesophases+for+structure+determination+by+macromolecular+crystallography.">Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography.</a> J. Vis. Exp. (67), e4001 (2012).</li></ol>

没有利益冲突的声明。

在这个视频文章中，我们已经演示了如何使用机器人自动设置在观结晶试验在96孔玻璃夹层板使用蛋白质充满油脂的中间相。在这项工作中所用的机器人专门设计的，包括一个正排量玻璃注射器的粘性和粘性的中间相的纳升体积的准确和可重复的交付，作为最初描述7。 机器人的准确度和精确度是很重要的功能。然而，这些特征的程度和规律，机器人的性能进行评估和校准。不用说， 机器人的性能应该被评估，而单 ​​独的板正在设立 。这是不恰当的假设，机器人将完美，离开了机器人在无人值守的情况下运行。周到和细心的经营者应在POS银行足球比赛要注意通过声音或东西时不正常工作，并立即纠正的外观。此外， 应该仔细检查每块板，经眼的均匀性以及内容尽快的密封板和在它之前放好结晶试验 。这应该只需要几秒钟来执行，并且可以做到，而下盘被加载。注意到，例如，特定井未正确填写可能突出一定的沉淀剂的分配尖端发生故障的事实。应交付的中间相被看作是不规则的，相关的故障项目，必须立即予以纠正。通过在组注意到这些问题，并作出相应的调整，将节省时间和材料，包括宝贵的脂质和膜蛋白。如果成像器是用来跟​​踪晶体生长，准确度和精确度也可以被监测，在成像过程中。例如，系统的问题，跌落或丸剂位置的成像表明，什么是不正确和，纠正行动上游某处的协议是必要的。 的利益，可靠的性能，因此机器人必须在定期校准和需要。校准应包括交付以及丸和沉淀放置在井的沉淀和中间相的音量。很明显，只要有可能，应校准使用卷和材料，类似那些在此期间所覆盖的质量控制工作进行的试验中，将用于完成。 同样重要的校准也是如此有库存的机器人零部件及用品供应充足。灾难和意外故障，堵塞和崩溃也时有发生。在这样的情况下，具有提供一个替换沉淀剂交付小费，例如，可能意味着一个极有价值的膜蛋白质制剂，因为它应该使用不被浪费。 的机器人的一些优点包括这样的事实，它将工作基本上连续的，它并没有患有或“抱怨”疲劳。机器人也可以用作不被视为人类友好的条件下，如在黑暗中，在控制的照明和环境条件，并且在极端的温度。在这篇文章中表现出的机器人〜20℃的环境条件下使用但是，也有蛋白质和项目，需要非环境温度，控制光11,12和氧化性或还原性环境13。所有这些都可以被照顾，相对容易，使用时的结晶机器人。 在早期的朱庇特视频文章中，我们演示了如何结晶实验的中尺度方法手动设置1。最小量的中间相可省去用手可靠有限公司by的视力，稳定霸道的人设立的试验。根据我们的经验，大量的中间相低至100 NL很容易处理。我们知道，一个实验室中手动分配的默认音量大约是40 NL。然而，相当小的体积可以使用机器人。另外，我们已经表明，机器人14可省去中间相体积低至550皮升。从这项工作进一步小型化的机器人，很明显，是可能的，如果实施的话，会导致宝贵的膜蛋白需要进行结晶试验的数量大量减少。 在这段视频中的文章，三种市售机器人被用来证明使用脂质介孔高通量膜蛋白结晶。其中第一个被开发的MS＆FB集团根据我们的经验，设立审判手动，如在朱庇特1712 1。这是仪器MENT我们有最熟悉和当前文章的大部分被用于其使用。另外两个机器人示范MS＆FB的时候写的文章，在这里的利益，完整性和相关的画面。所有这三个机器人使用非常相同的中间相分配系统，其中的本质特征是一种容积式玻璃注射器5,7。它们的不同之最显著方面的沉淀交付。机器人1和2可以同时免除沉淀剂为8个孔，在一个时间的结晶板的一个单列。相比之下，机器人分配沉淀所有96个解决方案，在一个单一的行动。机器人2是唯一的工具，用一次性沉淀分配技巧。有其优点和缺点，不同的乐器依赖于特定的应用相关，这些都不会在这里详细阐述。我只想说，所有这三个工作，并产生了晶体的我mbrane蛋白在中观法。 生物大分子晶体结构测定的整个过程的下一个步骤是收割和低温冷晶体板设立此视频文章中所描述的记录和处理X射线衍射。这些主题均包含在单独的朱庇特的这一系列文章中1,15。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 组件 </td></tr><tr><td> BRAYER（辊） </td><td> Fisher Scientific则</td><td> 50820937 </td><td>工具</td></tr><tr><td>气密性注射器</td><td>汉密尔顿</td><td> 81030 </td><td>工具</td></tr><tr><td>盖玻片</td><td>马林费尔德</td><td> 01029990911 </td><td>一次性</td></tr><tr><td>玻璃板</td><td>马林费尔德</td><td> 1527127092 </td><td>一次性</td></tr><tr><td>狮鹫LCP机器人</td><td>艺术罗宾斯</td><td> NA </td><td>工具</td></tr><tr><td> 中尺度机器人</td><td> Anachem /吉尔森</td><td> NA </td><td>工具</td></tr><tr><td>实验室笔记本</td><td>各个</td><td> NA </td><td>工具</td></tr><tr><td>蚊子LCP机器人</td><td> TTP LABTECH </td><td> NA </td><td>工具</td></tr><tr><td>穿孔双棍间隔带</td><td>桑德斯公司（孔打孔） </td><td>定制</td><td>一次性</td></tr><tr><td>沉淀剂解决方案</td><td>各个</td><td>各个</td><td>试剂</td></tr><tr><td>纯净水</td><td>密理博</td><td> NA </td><td>试剂</td></tr><tr><td>雨-X </td><td>壳牌汽车护理</td><td> 80199200 </td><td>试剂</td></tr><tr><td>注射器技巧</td><td>汉密尔顿</td><td> 7770-020（表22） </td><td>工具</td></tr><tr><td>组织</td><td>各个</td><td>各个</td><td>一次性</td></tr><tr><td>水瓶</td><td>各个</td><td> NA </td><td>试剂</td></tr></tbody></table>

use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases

膜蛋白的结构与功能的研究极大地受益于高分辨率的3-D结构的类型通过大分子的X射线晶体（MX）。 MX的一个重要成分，是理想的衍射高质量晶体的稳定供应。内的内消旋或脂质立方相（LCP）的膜蛋白结晶的方法是可用于膜蛋白结晶的几种方法之一。它使得使用的共连续的中间相，在晶体生长。作为一种方法，它有一些引人注目的成功后期，已经引起了人们的关注与兴趣使用它的许多研究小组现在。相关的挑战的方法之一是，主机中间相是非常粘稠，粘，让人联想起了厚厚的牙膏。因此，在一个可重复的方式小批量结晶井手动分配需要技巧，耐心和稳健的手。一种协议，用于做举ST开发的膜结构与功能的生物（MS＆FB）1〜3 组 。朱庇特视频文章描述的方法是1,4。 本手册的方法建立中尺度试验具有明显的优势与特色的应用程序，，如水晶优化和衍生。但它确实患有低通量的方法。在这里，我们展示了机器人表演观结晶试验的协议。一个机器人提供的优势，速度，准确性，精密化，小型化，能够长时间连续工作下可被视为恶劣条件下，如在黑暗中，在还原气氛中或高或低的温度下。 一个机器人观 ，正确使用时，可以大大提高生产力促进结晶，这是一个缓慢的步骤中的膜蛋白的结构和功能研究整体结构确定管道。 在这个视频文章中，我们演示了如何使用三种市售的机器人，可以免除的粘性和粘性的中间相不可或缺的观 crystallogenesis，。第一个机器人开发在MS＆FB 5,6组。另外两个最近成为这里包括的完整性。 在本文中涉及的协议的概述示于图1，所有的操作均在室温（〜20℃）的环境条件下进行。

Watch this Scientific Journal Video about Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases at JoVE.com

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

本文中描述了一种机器人的方法，在使用大分子X-射线晶体结构的测定中使用的脂质中间相为高通量膜蛋白结晶。 3个机器人能处理粘性和粘蛋白载货中间相积分的方法进行了介绍。

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

use-robot-for-high-throughput-crystallization-membrane-proteins

Research

JoVE Journal

Biology

17.5K 次观看.  Trinity College Dublin. 本文中描述了一种机器人的方法，在使用大分子X-射线晶体结构的测定中使用的脂质中间相为高通量膜蛋白结晶。 3个机器人能处理粘性和粘蛋白载货中间相积分的方法进行了介绍。

视频: Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

科研

生物学

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

评估电子晶体结构生物学研究的小膜蛋白的二维结晶试验

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

X射线晶体学的蛋白质结晶

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...