有很多谁促成了这一工作，大部分是从卡弗里膜的结构与功能的生物集团，无论是过去和现在的成员。 ，尤其是景泉潭和约瑟夫·利昂，我们致以最热烈的感谢和赞赏。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成本和玛丽·居里行动（CM0902和PIEF-GA-2009 -235612）。

1。实验室设置捕前<ol><li>在收获的准备，填补了干性发泡杜瓦用液态氮，并把它旁边的收获是发生在显微镜下。 </li><li>被淹没的存储冰球，打开结束了，里面的泡沫杜瓦在液态氮中，并允许它完全冷却下来。 </li><li>固定安装的尺寸相匹配的晶体上的磁力棒（ 图5）将收获微。这是重要的手头上有一些备用磁性棒预装微坐骑，以配合的情况下，这是必要的收获几个晶体从一个大丸剂。备用魔杖应该是可以在任何时候进行。 </li><li>将微管，技巧和沉淀的解决方案，用于晶体生长的收获显微镜。它可能需要以覆盖中间相和，以防止干燥的井被打开时。 </li><li>在板凳上有一个笔记本和一支笔，关闭和/或在计算机上打开。这些将被用来记录有关的质量，外观，位置，存储圆盘号码，等，结晶，因为他们被采伐，低温冷却，并放置在存储的观测。 </li><li>如果一个助理可以帮助与收获的人一定要了解清楚的协议之后，将各个步骤的顺序会发生，和他们在整个过程中的作用。 </li></ol>所有的材料和设备在我们的下一个任务是: 2。确定板和样品中的晶体<ol><li>最简单和最直接的方法寻找收获的晶体是手使用正常和交叉的偏振光显微镜检查井。在显微镜下可以帮助调整照明光线强度与定位的结晶。 </li><li>另外，成像板自动筛选与正常和交叉极化D灯，可以用来寻找晶体。 </li><li>评估用眼睛记录的数字图像在计算机显示器上。 </li><li>清楚地标明这些井的大小，质量和位置的中间相在笔记本电脑或一台计算机上的晶体在晶体的收获和记录意见。 </li><li>删除板中晶体的热像仪的收获。 </li></ol> 3。打开以及与立方中间相。方法1 由内消旋方法，其中晶体生长的板是玻璃夹心板（ 图2）。为了访问的中间相，其中的结晶，它是必要的，以打开孔。这是通过使用一个玻璃切割工具切割的上部的盖片玻璃密封的井可以被删除。 切割和取出盖玻片的结晶，有几种方法。使用的是出于O型f中间相的晶体生长。这可以是很粘稠的和粘性的立方相（ 图3A）或更多的流体变型中，海绵相（ 图3B）。在这个视频文章中，我们将展示如何打开井和收获这些托管材料的晶体。 <ol><li>放置在舞台上的光显微镜的玻璃夹层结晶板。 </li><li>使用的玻璃切割工具得分盖玻片轻轻带两​​个同心圆，位于上方的隔板和外面的周边的阱。有了一个新的刀具，需要施加的压力降至​​最低得分。更换时所需的压力得分甚至小幅增加一个新的工具，这通常发生在开放10口井。 </li><li>分手两个得分圈之间的空间中的玻璃释放的内盖片玻璃的目的。这会产生大量的玻璃碎片和尘埃。清除他们用湿纸巾。 </li><li>抓住用细尖镊子和倾斜距离和关闭以及删除释放的玻璃罩。在这种情况下，立方相仍然卡住，以代替在底板上的阱。 </li><li>放大得到一个更清晰的视野立方中间相准备收获的晶体使用。 </li></ol> 4。打开一个井三次中间相。方法2 <ol><li>使用刀具得分直的平行线，在盖玻片一侧的阱和延伸横跨井本身的玻璃。这使得更容易镊子访问和去除盖玻片。 </li></ol>在这个特殊的示范，使盖玻片粘垫片表面裂缝的玻璃罩。盖玻片转移的过程中，在适当位置，从立方相分离和沉淀剂。当玻璃罩取消一些的沉淀"。我们只剩下裸露bOLUS的中间相，没有任何沉淀。随即，丸，用微量吸移管，以防止中间相从干燥和经历相变，这可能会损坏晶体上加1微升新鲜沉淀剂。丸是现在随时可以使用的晶体收获。 5。打开井与海绵相。未成功海绵相是不够宽容，因为它的流动能力。如果该流的查询结果在海绵中相接触的周边的阱毛细将抽出的中间相，将丢失的结晶。发生这种情况的一个例子是在这个视频剪辑。 <ol><li>放大的海绵上的相和正常和正交偏振光之间来回切换，定位在海绵相的晶体。 </li><li>在开放的好成绩，并削减盖玻片，在第3节的准备。在这个过程中，在盖片玻璃裂纹。在这方面所做的尝试ING打开以及沉淀剂的变化的方向的裂纹，并最终它是在与隔板接触。随着沉淀"的海绵相及其晶体，丢失的货物。 </li></ol>在这个特定序列，在显微镜上的偏振片没有完全划线，并晶体明亮的物体，在同一时间可以被看作是该井和其内容保持可见。 6。打开井与海绵相。顺利<ol><li>缩小在海绵阶段和拣选晶体在正常和正交偏振光。 </li><li>分数，剪切和删除一个部分的盖玻片覆盖的良好，在4.1节。不完全交叉的偏振光被用于跟踪的晶体。 </li><li>通过开放的玻璃罩和入井，直到它刚好接触到沉淀剂溶液一块干纸巾介绍。通过毛细作用带走的解决方案小心LY，直到它几乎全部消失了，然后取下的组织。这将导致剩余的沉淀剂和海绵相，与晶体仍然到位，回缩根据盖玻片。 </li><li>分数，剪切和删除与镊子盖玻片的其余部分，如在4.1节。在这种情况下，海绵相分裂;一些保持在阱和一些枝盖玻片。该晶体是在盖玻片上的丸。因为很少有沉淀剂本，海绵相开始经过相变可能是由于干燥。这可以看出，作为环的双折射迁移丸剂朝向中心。立即加入沉淀剂，丸剂，以制止过渡。丸是现在准备收获的晶体使用。 </li></ol> 7。收获和低温冷晶体立方相<ol><li>回正常和正交偏振光之间来回定位在公开以及在立方相丸剂晶体。在t可以看出，在正交偏振光的的立方相丸剂与他的视频序列4双折射晶体。 </li><li>使用安装冷冻循环（ 图5），探讨新暴露的中间相对于晶体，捞出，然后令它们的晶体，在低温循环，放入液氮杜瓦后， 立即收获。理想的情况下，收获和冷却单元应该发生在一个连续和快速运动。尽可能少附着中间相应尽可能与晶体收获。在我们的经验中，不需要冷冻保护与细观生长的晶体。 </li><li>因为它是不可能的晶体在低温循环收获后立即检查中间相丸用于收获验证的晶体不再存在，这表明它是收获成功的。 </li></ol> 8。采收和低温冷晶体的海绵相<ol><li>回正常和正交偏振光之间来回定位在海绵中的结晶相在公开以及丸剂。在这个视频序列的许多双折射晶体，可以看出，在正交偏振光下的丸剂。 </li><li>使用安装低温回路（ 图5），捞出晶体的海绵相，并令它们，载或上cryoloop，投入液氮杜瓦后， 立即收获。理想的情况下，立方相的收获，低温冷却过程应该立即发生的晶体收获尽可能短的时间内可能经过的实际收获事件和陷入液氮。尽可能少附着海绵相应尽可能与晶体收获。如前所述，不需要冷冻保护与细观生长的晶体。 </li></ol> 9。在杜瓦存储晶体<ol><li>使安装循环转换成液态Ni含氮将其放置在一个存储在泡沫杜瓦冰球的保持槽。完成所有的操作循环，磁性棒的顶部和冰球浸没在液氮中。 </li><li>记录的位置和细节的收获和低温冷却结晶中的笔记本电脑和/或计算机上。 </li><li>当冰球在泡沫杜瓦是充满收获的一天填妥之过户成一个搁置冰球冰球在存储或传输杜瓦持有人充满液态氮。晶体可运交通杜瓦瓶收集衍射数据的同步辐射设施。 </li></ol> 10。代表性的成果这里展示的是收获和低温冷却练习的目标转移从托管到冷冻循环的中间相的晶体，玻璃化的环形晶体，并把它储存在液氮的杜瓦。理想的情况是收获和c坂冷却完成在这样一种方式，衍射的晶体质量的过程中被保留。尽可能少的中间相应尽可能与晶体收获。这是为了使定位的晶体和围绕它的X-射线束，少得多有挑战性，加快低温冷却下玻璃化的视图，并尽量减少干扰背景散射衍射数据收集期间，从中间相。低温冷却的样品的晶体能够和不能够被看到的一些例子示于图6。凡晶体不能看到它的眼睛通常是必要的，以便找到的晶体和数据收集27中的光束，使其中心诉诸衍射光栅。 <img src="/files/ftp_upload/4001/4001fig1.jpg" alt="图1" /> 图1的示意图示出的脂质双分子层，并在其上的生物膜芳ë位于的各种蛋白质。 <img src="/files/ftp_upload/4001/4001fig2.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。 <img src="/files/ftp_upload/4001/4001fig3.jpg" alt="图3" /> 图3。膜蛋白的晶体生长在脂质中间相。 H.细菌视紫红质晶体的立方相杆菌 。B.海绵相的维生素B12受体/转运，BtuB的含有晶体，从大肠杆菌大肠杆菌 。从参考文献25。立方和海绵阶段有对比APPEA的rances是显而易见的通过比较面板A和B A是极其粘稠的，并保持其原来的形状的立方相。它不流动。这是特别明显的，在边缘处的中间相的大丸剂，具有粗糙的外观。合同，的海绵相是相当少的粘性和不流。因此，海绵相不保留其原来的形状，是典型的边缘平滑。沉淀剂，其中包括聚醚胺，2 -甲基-2,4 -戊二醇丙氧基化物，丁二醇，己二醇，季戊四醇，聚乙二醇400，可能会导致在一个过渡海绵从立方相4,26。 <img src="/files/ftp_upload/4001/4001fig4.jpg" alt="图4" /> 图4。流程图总结了在生产，收获和低温冷观种植的膜蛋白晶体（A）所涉及的步骤。只有那些红色虚线包围的步骤线，和（B）中，详细描述在此JOVE文章覆盖。 A组是从参考文献3。 <a href="http://www.jove.com/files/ftp_upload/4001/4001fig4large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/4001/4001fig5.jpg" alt="图5" /> 图5。举行的PIN码在地方上的磁力棒安装在一个空的冷冻循环。扩展的意见引脚（A）和微卡口（B）的收获和低温冷却这一重要工具，如图所示。接环B 中的微空循环结束时的直径为30μm。虽然没有广泛测试了其他类型的循环中，我们发现循环显示，MiTeGen面心立方和海绵阶段的工作。 <img src="/files/ftp_upload/4001/4001fig6.jpg" alt="图6" /> 图6。收获和低温冷却的膜蛋白晶体在低温循环与同步辐射光束线在显微镜观察A，B，例如收获的晶体（caa3细胞色素氧化酶34（A），甘油二酯激酶，DgkA （B））的结晶（蓝色箭头）通过低温冷却的中间相上的冷冻回路。C.实施例，其中收获晶体上的冷冻回路中的低温冷却的中间相是不可见的可见。用红色箭头标识的尖端循环。秉承低温冷却的中间相确定了一个蓝色的箭头。

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A., Huang, X. P., Carroll, F. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+human+kappa-opioid+receptor+in+complex+with+JDTic.">Structure of the human kappa-opioid receptor in complex with JDTic.</a> Nature. 485, 327-332 (2012).</li><li>Cherezov, V., Abola, E., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+structure+determination+of+GPCRs,+a+membrane+protein+family+with+high+potential+as+pharmaceutical+targets.">Recent progress in the structure determination of GPCRs, a membrane protein family with high potential as pharmaceutical targets.</a> Methods Mol. Biol. 654, 141-168 (2010).</li><li>Liu, W., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+membrane+proteins+in+lipidic+mesophases.">Crystallization of membrane proteins in lipidic mesophases.</a> J. Vis. Exp. , (2011).</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=Nano-volume+plates+with+excellent+optical+properties+for+fast,+inexpensive+crystallization+screening+of+membrane+proteins.">Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins.</a> J. Appl. Cryst. 36, 1372-1377 (2003).</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. Cryst. 39, 604-606 (2006).</li><li>W&#246;hri, A. B., Johansson, L. C., Wadsten-Hindrichsen, P., Wahlgren, W. Y., Fischer, G., Horsefield, R., Katona, G., Nyblom, M., Oberg, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Lipidic-Sponge+Phase+Screen+for+Membrane+Protein+Crystallization.">A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization.</a> Structure. 16, 1003-1009 (2008).</li><li>Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rastering+strategy+for+screening+and+centring+of+microcrystal+samples+of+human+membrane+proteins+with+a+sub-10+microm+size+X-ray+synchrotron.">Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 microm size X-ray synchrotron.</a> 6, 587-597 (2009).</li><li>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=A+lipid's+eye+view+of+membrane+protein+crystallization+in+mesophases.">A lipid's eye view of membrane protein crystallization in mesophases.</a> Curr. 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Exp. , e1712 (2010).</li><li>Li, D., Boland, C., 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=Use+of+a+robot+for+high-throughput+crystallization+of+membrane+proteins+in+lipidic+mesophase.">Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophase.</a> J. Vis. Exp. , e4000 (2012).</li><li>Lyons, J. A., Aragao, D., Slattery, O., Pisliakov, A. V., Soulimane, T., 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=Structure+insights+into+electron+transfer+in+caa(3)-type+cytochrome+oxidase.">Structure insights into electron transfer in caa(3)-type cytochrome oxidase.</a> Nature. 10, (2012).</li></ol>

没有利益冲突的声明。

在这个视频文章中，我们演示了如何脂质中间相的晶体生长，收获和低温冷却衍射数据收集的准备，最终确定的结构。主机中间相的粘性和粘立方相或更流畅的海绵相4。玻璃夹层板被打开，如何晶体的收获非常依赖于中间相类型。因此重要的是知道这两个1处理的时间提前。托管脂质的身份和所用的沉淀剂在这方面是重要的，和大丸剂中的结晶化的中间相的物理外观，以及可以用来告诉他们分开（ 图3）。在这篇文章中，收获从两者中间相类型进行了说明。 收获小晶体从的脂质中间相在玻璃夹层板是一个艰苦的过程，需要时间，技巧，experience，耐心和稳健的手。重要的是要预留适量的收获，并设立了实验室，使所有的用品和设备在手提前。第二协助收割的人是没有必要的，但建议使用。这个人可以帮助提供预显着的板，做收获的个人以及低温冷却安装到存储冰球收获晶体的循环。助理也发挥了重要的支撑作用在记录观察在收获的晶体衍射数据收集过程中，这可能是至关重要的。在一个助手的情况下，语音激活音频记录装置可以有利地使用的文档。 在这篇文章中描述的协议将有助于启动和运行的浏览器与晶体收获。然而，重要的是要理解，该过程并不简单，，的PRActice之前，需要启动到收获宝贵的膜蛋白晶体。因此，建议先测试板不是特别宝贵的蛋白质晶体进行实验。这将提供了宝贵的经验，在切割玻璃的新手，取出玻璃碎片，抬起玻璃罩从中间相，采用偏光显微镜功能晶体和跟踪他们在收获，最后处理不同类型的介孔从他们和收获。中间相的质地，并且通过扩展的难易程度，晶体可以收获，不随时间变化，在结晶过程中。因此重要的是练习价值较低的晶体，但是与那些已经为更有价值的相同的条件下生长的收获。它可以由消旋或脂质立方相的方法28和晶体生长的溶菌酶和thaumatin应应该考虑这些ERED等效的方式获得的材料和方法的熟悉。人们还应该考虑与无蛋白质的中间相，首先要学会它的变幻莫测。 这里展示的程序都在一个舒适的20°C或左右完成。这是可能的内消旋的方法中，在较低的温度下晶体生长。因此，油酸单甘油酯作为宿主脂质在亚稳相状态，可以使用在4℃下1,2,29,30。一种替代方法是使用合理设计的7.9 MAG低温晶化31。我们通常做低温crysallogenesis的，具有一定的膜蛋白指标。在这种情况下，晶体生长和收获在散步，在4℃冰箱中。在这样的条件下工作，有其自身的挑战，其中最重要的是需要温暖和舒适的服装。 使用大分子晶体结构测定的整体过程中的下一步骤的tallography是收获和管理单元冷却在这篇文章中所表现出的对晶体的衍射数据收集。， 在细观生长的晶体通常小。然而，有用的衍射数据收集有可能与具有最大尺寸为20μm的9的晶体。为了这个目的，使用微束同步辐射X-辐射，是一个单独的JOVE这一系列中的相关文章32,33的焦点。

<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>弯曲的镊子</td><td>西格玛</td><td> F4142 </td><td>工具</td></tr><tr><td>一次性吸头</td><td>吉尔森</td><td>各个</td><td>一次性</td></tr><tr><td>泡沫杜瓦</td><td> Spearlab </td><td> FD-500 </td><td>工具</td></tr><tr><td>玻璃和金属废物箱</td><td>丹尼尔斯医疗保健</td><td> DD479OL </td><td>工具</td></tr><tr><td>收获的循环</td><td> MiTeGen </td><td>各个</td><td>工具</td></tr><tr><td>收获显微镜</td><td>尼康</td><td> SMZ1500 </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>磁推按钮样品装载棒</td><td>汉普顿研究/分子尺寸</td><td> HR4-729/MD7-411 </td><td>工具</td></tr><tr><td>原始顽童（只使用ALS式机器人） </td><td>水晶定位系统</td><td> CP-111-035 </td><td>工具</td></tr><tr><td>加样器</td><td>吉尔森</td><td>各个</td><td>工具</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>帕克特冷冻通</td><td>水晶定位系统</td><td> CP-111-030 </td><td>工具</td></tr><tr><td>帕克搁置运输甘蔗（原ALS式）与弯曲的把手和锁杆</td><td>水晶定位系统</td><td> CP-111-029 </td><td>工具</td></tr><tr><td>纯净水</td><td>密理博</td><td></td><td>试剂</td></tr><tr><td>安全护目镜</td><tD&gt;各种</td><td> NA </td><td>工具</td></tr><tr><td>样品针基地 - 磁（铜） </td><td>水晶定位系统</td><td> CP-111-015 </td><td>工具</td></tr><tr><td>航运杜瓦</td><td>泰勒 - 沃顿商学院</td><td> CX100 </td><td>工具</td></tr><tr><td>组织</td><td> NA </td><td> NA </td><td>一次性</td></tr><tr><td>碳化钨玻璃切割机（TCT划线） </td><td>银线工具（英国Yeovil） </td><td> 633657 </td><td>工具</td></tr></tbody></table>

harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography

了解蛋白质的功能在机械水平的一个重要途径是，有靶蛋白的结构，最好是在原子级分辨率。目前，只有一个施加到完整的膜蛋白（ 图1），和它们形成的复合物的方式来捕捉此类信息，并且该方法是大分子X-射线结晶器（MX）。要做到MX衍射质量晶体的需要，在膜蛋白的情况下，不容易形成。膜蛋白，包括利用，脂质介孔，特别是的立方和海绵阶段1-5，结晶的方法获得了相当的关注的晚期由于取得了G蛋白偶联受体领域6-21（ <a href="http://www.mpdb.tcd.ie" target="_blank">WWW 。mpdb.tcd.ie</a> ）。但是，方法，以下简称为中观或脂质立方相法，配有其自身的技术的挑战。这些出现时，一部分原因是由于脂质中间相性质，在其中，这往往是微晶体，晶体生长的一般粘性和粘性。操作晶体的结果变得困难，特别是在收获22,23。问题出现时太在收获的步骤之前要求的玻璃夹层板，其中晶体的生长（ 图2）24,25被打开以暴露中间相丸剂，其中的结晶，收获，低温冷却和最终X -射线衍射数据的收集。 立方和的海绵中间相变体（ 图3）晶体收获都必须有深刻的不同的流变性4,26。立方相的粘性和粘类似于一个厚厚的牙膏。与此相反，海绵相多个流体流动具有鲜明的倾向。因此，不同的方法来打开结晶井containing的晶体生长在立方相和海绵相被称为作确实需要不同的方法收获​​从两个中间相类型的晶体。协议只是做，已细化和落实在膜结构与功能的生物集团（MS＆FB），并详细介绍了在此朱庇特的文章（ 图4）。实施例中给出的情况，在结晶成功收获和低温冷却的。我们还提供例子的情况下，出现问题，导致无法挽回的损失的晶体，并说明如何避免这些问题都可以。在这篇文章的浏览器提供了一步一步的指示，打开玻璃夹层的结晶井，收获和低温冷却的膜蛋白晶体生长立方和海绵阶段。

Watch this Scientific Journal Video about Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography at JoVE.co

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

本文所描述的过程中实现卡弗里膜的结构和功能的生物集团收获和低温冷膜蛋白晶体生长大分子的X射线晶体结构测定中使用的油脂的立方和海绵阶段。

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 ...

harvesting-cryo-cooling-crystals-membrane-proteins-grown-lipidic

Research

JoVE Journal

Biology

24.8K 次观看.  Trinity College Dublin. 本文所描述的过程中实现卡弗里膜的结构和功能的生物集团收获和低温冷膜蛋白晶体生长大分子的X射线晶体结构测定中使用的油脂的立方和海绵阶段。

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

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

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

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 ...

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 ...

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

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

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 ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.