This work was supported by grant NIH R01 GM097204 (APV and JDH). Funding for JHM was supplied by an HHMI Undergraduate Research Summer Fellowship.

注:概念框架或工作流的示意图在图1中被描述以供参考。该协议可以被分解成四个阶段:计算或基于序列的预测，蛋白质表达和纯化，生化特性和结晶。示出的实施例分析蘑菇时SD2域和/或蘑菇时摇滚络合物，但可以与任何蛋白质被利用。 1.使用建立基于Web的工具来生成卷曲螺旋域边界的计算预测<ol><li>收集的进化多样化序列同系物。 <ol><li>转到www.uniprot.org并在上部搜索栏中输入蘑菇时。 </li><li>选择序列通过检查旁边的登录号框下载。用星序列指示由UNIPROT审查序列并且通常更加可靠。请注意，以确保所有的序列完整，准确。 </li><li>保存选定S通过单击下载按钮equences。 </li><li>对齐蘑菇时序列使用的Clustal欧米茄21（http://www.ebi.ac.uk/Tools/msa/clustalo/）集合。点击"浏览"按钮，选择与蘑菇时序列的文件，然后按提交。 </li><li>校准完成后，点击"下载对齐文件"按钮。 </li><li>打开使用Jalview在1.1.5中产生的多重序列比对（文件|输入对齐|从文件）。在新的窗口对齐，通过颜色标识（颜色|同一性百分比）。 </li></ol></li><li>计算二级结构预测，柔性的或无序的序列的预测，和感兴趣的蛋白质（多个）内的预测的卷曲螺旋区域。 <ol><li>去http://gpcr.biocomp.unibo.it/cgi/predictors/cc/pred_cchmm.cgi，计算出卷曲螺旋预测。复制和序列到序列糊盒并点击提交。 注:此分析可能需要几个小时COMP勒特。因为蘑菇时序列是相当大的，所述序列可能需要被分成少于1000个氨基酸的块以适合Web服务器的尺寸限制内。当分析Shrm2，则建议的C-末端1000氨基酸被用作该部分包含这是已知的包含卷曲螺旋区域SHRM SD2域。当重复该分析与其他蛋白质，建议可能时使用的全长蛋白质序列。如果这不是一个选项，使用最大的片段可能或解剖基于已知生化或功能性数据的顺序。 </li><li>去http://gpcr.biocomp.unibo.it/cgi/predictors/cc/pred_cchmm.cgi，计算出卷曲螺旋预测。复制和序列到序列糊盒并点击提交。 </li></ol></li><li>结合步骤1.1至1.2.2结果到蘑菇时序列的单一综合注释。 注:强烈鼓励还包括任何及所有有关我载文信息可以从文献或约在此阶段的蛋白质的功能或纯化其他来源收集。 </li><li>采用这种综合注解序列，预测域边界（或一系列可能的边界）的蛋白质，试图最大化养护和同时最小化紊乱或柔性序列的量的预测的结构特征。如果公知的，所得到的蛋白应保留感兴趣的功能特性。 </li></ol> 2.表达和纯化蛋白质在第1节确定的域边界注意:本节的目标是使用了一系列快速，轻松地量化分析来筛选在第1节产生的假想域边界。 <ol><li>使用标准的分子生物学技术，生成与他的10 -mRuby2-XH2质粒内帧所需的编码序列的表达质粒（参见图3为矢量地图ND更多详细信息）。 </li><li>测试表达水平使用BL21（DE3） 大肠杆菌或其他合适的表达菌株在自身诱导介质1在室温下18-24小时，各个表达质粒。 <ol><li>变换之后，与细胞来或者使用健壮转化方案的说明，如这里所述一个（https://www.addgene.org/plasmid-protocols/bacterial表达质粒以及一个空质粒控制到BL21（DE3）中-转型/）。 </li><li>从新鲜的转化板，挑取单个菌落并在LB培养基过夜37°C（LB）培养基上生长5毫升发酵剂培养物与34微克/毫升卡那霉素。 </li><li>第二天，沉淀培养并用新鲜的LB中洗涤沉淀</li><li>起始培养可添加50毫升自身诱导培养基用34微克/毫升卡那霉素。允许培养物以在室温或37℃生长至饱和。通常情况下，生长〜18-24小时。 </li><li>颗粒细胞S和冷冻所得的细胞沉淀在-80℃下无限期之前纯化。 </li><li>比较来自每个表达菌株和通过SDS-PAGE的空载体对照全细胞提取物，以确定最有效地产生所需的融合蛋白的菌株。为他的10 -mRuby2-蘑菇时SD2融合蛋白，在180 V代表〜50分钟或直至染料前沿到达凝胶22的底部运行的10％SDS-PAGE凝胶。 </li><li>用考马斯蓝染色的凝胶，以每种菌株23内可视总细胞蛋白质。 </li></ol></li><li>对那个成功表达mRuby2融合蛋白每株初始亲和层析一步。通常情况下，执行净化步骤2.3.2-2.3.7在室温下，除非该蛋白质将改变的条件如在4℃纯化受益。 <ol><li>从步骤2.2.5解冻细胞团。 </li><li>重悬每个细胞沉淀在1.5ml裂解缓冲液（10％甘油，500毫摩尔NaCl，40的mM咪唑，20毫摩尔Tris pH8.0的，1毫β-巯基乙醇）。补充有蛋白酶抑制剂适当的裂解缓冲液。 </li><li>添加30微升10毫克/毫升溶菌酶，在室温下孵育20分钟。使用超声处理下列仪器的指示。 </li><li>转移到1.5ml试管并离心在台式离心机30分钟在14000 xg离心以沉淀不溶的物质或未溶解的细胞。节省上清液和沉淀通过SDS-PAGE随后的分析。 </li><li>执行镍亲和纯化，孵育用100μl的Ni-NTA珠的可溶级分。孵育可溶性裂解物珠粒5-10分钟，翻转数次以混合珠。 </li><li>离心以800×g离心30秒在台式离心机以沉淀珠。用裂解液洗涤沉淀3-5次清理掉非特异性的污染物按照此。 </li><li>从用裂解缓冲液珠洗脱红宝石融合蛋白补充有11M咪唑。 </li><li>使用SDS-PAGE来比较他-mRuby2-蘑菇时蛋白质的行为，在"快速和肮脏的"净化以上。 </li></ol></li></ol> 3.表征蛋白质样品识别那些具有有利的性能<ol><li>使用分光光度计组在280nm处测量的Ruby融合蛋白的浓度，然后通过加载融合蛋白的1-5微克到天然PAGE凝胶24评估样品的均匀性。在4℃下在175五运行10％非变性PAGE凝胶140分钟<ol><li>图像使用配备可视化的荧光，观察其中mRuby标记融合在该测定中迁移的成像器的非变性PAGE。 注意:小心观察到，被卡在以及这种蛋白质很可能聚集的融合蛋白。如果一个荧光成像器不可用，经常可以形象化和Ruby的图像浓缩样本下一个黑光或用UV光盒标签蛋白。 </li></ol></li><li>执行有限的蛋白水解识别稳定折叠域。孵育95微升红宝石SHRM SD2融合在〜1毫克/毫升用5微升0.025％枯草杆菌A的<ol><li>样在时间点反应0,0.5，2，5，15，60，和120分钟，从每个时间点的反应去除10微升，并使用15％丙烯酰胺凝胶可视化通过SDS-PAGE的反应的进程。 </li><li>用考马斯蓝染色在步骤2.2.7和评估蛋白酶抗性的物种是否可以被识别。 </li></ol></li><li>从集成净化效率，行为本土PAGE和有限的蛋白水解对部分纯化mRuby2，蘑菇时融合蛋白进入数据在溶液中这些蛋白的整体行为进行了全面评估。 <ol><li> （可选）如果合适的功能性试验，则需要检查的活动在这一点上。 </li></ol></li></ol> 4.生产卷曲螺旋SD2的高品质晶体域中的蘑菇时注:第4.1节中的所有步骤，在室温下执行，除非蛋白质将从净化在不同的温度中受益，一般为4℃。 <ol><li>使用50ml的生长作为表达和纯化潜在的粗略估计，执行mRuby2-蘑菇时SD2的大规模表达与实现5-20毫克完全纯化的样品的目标。典型地2升培养物提供了足够的原料。后在8000 xg离心10分钟，生长离心沉淀细胞。 <ol><li>重悬从作为之前在裂解缓冲液的2升生长沉淀，用约2ml每克冷冻的细胞沉淀的裂解如果用溶菌酶进行裂解。或者，如果使用悬浮在〜8毫升/ g的颗粒均质或法国媒体。在30000 XG 30分钟，小球通过离心细胞碎片。 </li><li>批次用10ml的Ni-NTA树脂的结合，从4.1.1可溶部分。倒入适当的重力柱状并允许未结合的蛋白流掉。 </li><li>洗涤树脂3-5次用40ml裂解缓冲液中，随后用补充至1M NaCl的裂解缓冲液洗涤。 </li><li>洗涤树脂用40ml补充有80 mM咪唑的裂解缓冲液。 </li><li>用40毫升裂解液的补充，以1M咪唑洗脱红宝石蘑菇时融合蛋白。手动分馏洗脱的蛋白到4-10毫升的级分。 </li><li>确认含有用10％的SDS-PAGE红宝石蘑菇时融合蛋白的级分。 </li><li>透析适当的级分（通常是馏分2-4）到8％的甘油，250mM的氯化钠，15mM的咪唑，20毫摩尔Tris pH8.0的，1毫β-ME，使用6-8 kDa的MWCO的透析管。加入1毫克TEV蛋白酶为每25-50毫克融合蛋白。在室温下缓慢搅拌下透析过夜。 </li><li>重复镍亲和纯化步骤4.1.2。到4.1.6。该蘑菇时SD2域现在应该留在未结合部分，而他的10 -mRuby2将继续必然树脂和将在洗脱级分中找到。确认用考马斯蓝此使用12％SDS-PAGE染色。 </li><li>透析含有蘑菇时SD2域馏分成8％甘油，100mM NaCl的20毫摩尔Tris pH值8.0，5mM的β-巯基乙醇缓冲液过夜。 </li><li>执行使用FPLC的阴离子交换层析25，并与来自0.1-1.0 M氯化钠一个NaCl梯度洗脱。分析用12％的SDS-PAGE的级分。 </li><li>使用旋转浓缩器（10千道尔顿MWCO或取决于所研究的蛋白质更高），集中的峰值馏分〜0毫克/毫升，然后执行大小排阻层析26，通过12％的SDS-PAGE分析峰级分。 </li><li>池峰级分并透析入的20mM Tris pH值8.0，50 mM氯化钠，2％甘油，1mMβ-ME，和浓缩至10mg结晶/毫升之前。 选择性:在此步骤中，除去5微升样品在浓度1，2，5和10毫克/毫升浓缩样品的过程中。运行每个样品的天然PAGE上样2-5微升该步骤期间看样品的行为。 </li></ol></li><li>筛选的12的条件的小阵列，以确定结晶试验的最佳蛋白浓度。这些条件的组合物在图7中说明。 <ol><li>使用24孔坐滴结晶盘中，使用蒸汽扩散法，移液进行结晶试验将500μl每种12条件成单独的孔中，随后逐滴最初含有孔溶液的1微升并在盖玻片1μl的蛋白质样品的。请参阅27对这项技术的更多信息。 </li><li>赶紧用密封透明胶带托盘和托盘移动到合适的温度控制的环境中，是免费的震动。使用可改变温度，但4℃，16℃，20℃是相当普遍。 </li><li>检查托盘滴在接下来的3天使用以高达100X放大率的显微镜AYS。在3天的期间结束，得分每个液滴含有无沉淀（清），光沉淀，重沉淀（棕色），或晶体。合适的蛋白质浓度应含有不超过6重沉淀。 </li><li>使用在4.2中确定的蛋白浓度，屏幕广泛市售结晶屏幕。使用液体处理或结晶机器人大大加快这一过程，而在滴也最小化误差。它需要少得多的蛋白，以及，允许用户使用单个样品筛选多个条件。 </li><li>通过系统地改变使用与以前的24孔筛盘的结晶下降中每个变量的提高，从广阔的屏幕识别的初始条件。 </li></ol></li></ol>

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The authors have nothing to disclose.

这里所描述的协议旨在帮助用户识别大型卷曲螺旋蛋白中域边界，以方便他们的结晶。该协议依赖于多种由计算预测和其他来源的数据的整体并入以产生一系列可能的域的边界。这些后跟一组生化实验这是快速和廉价的，并且被用来进一步缩小这些初始假设。使用这种方法，用户可以很快消除是不可取的潜在的蛋白质片段，并专注于更好的候选人更多的关注，从而提高获得晶体的前景。 有许多重要的步骤此技术中，然而，没有用于生产结晶作为初始设定假想域边界的发展至关重要。此步骤采用了各种计算方法，以及信息的ob从文献或功能性数据时可用tained。应当小心，以避免使用该策略，因为目前的地方到一个量化的置信值附加到任何预测的无法立即磨练下至单个"最佳"的解决方案。相反，它是最好的作为一个方法来快速识别一小部分，需要加以实验验证可能域边界。 可与该协议被分析卷曲螺旋蛋白质的特性是相当广阔的。从计算的角度来看，卷曲螺旋预测的强度是有限的下面20个氨基酸，并如在步骤1.2.1所述，卷曲螺旋区域大于1000氨基酸需要由DISOPRED被分成多个部分以进行分析。我们认为是暂时的，因为它是由网络服务器和征收的方法在未来的升级可能会改变这种后来限制。随后的生化分析然而，可以承受以各种方式。首先，内部循环或过敏的蛋白酶可能使样品区域显得比它实际上是不太稳定的。已切割环或通过蛋白酶以其他方式缺口稳定蛋白仍应给上变性PAGE一个稳定的外观，这就是为什么它建议用户探索两种策略。本地PAGE可能是困难的一些蛋白质，但是，无论是因为他们是很长，慢慢迁移到凝胶或因为他们的特殊费用可能会让他们走出凝胶反方向跑完全。在此情况下，可能是有益的探索不同的缓冲条件的天然PAGE凝胶上的系统。 而这里介绍的工作的重点是大含卷曲螺旋蛋白，该协议可用于非常少，几乎适应任何蛋白质。主除了为球状蛋白质将是除了域预测软件如pDomTHREADER 28 &lt;的/ SUP&gt;或DomPred 29寻找域边界，和三级结构的预测，如Phyre2 30以增强序列分析的预测能力。此外，还有一些可用于执行二级结构预测和无序的预测许多算法，以及额外的算法包含可能是有帮助的。球状蛋白质往往具有酶活性或其它功能读数，将目标选择过程中提供重要的附加信息。此外，中等或高通量的技术可以并入适当。例如，对于1-2毫升培养物和金属亲和纯化在96孔格式便宜的系统是容易得到31。最后，用于设置大量使用最少的样品结晶实验中使用的机器人的是变例程，但机器人系统的高效运行的前提是充分表征的蛋白质样品的行为。加成人修改本协议可能包括多种采样亲和标签。许多不同的荧光标签可用，或者的His-，GST-，Thioredoxin-，Strep-和MBP-几十个可用选项中如果不需要或有用荧光标记。 当执行此过程，用户应注意的是，mRuby2标签可能对用户的兴趣蛋白质的效果。使用对各种不同融合物该标签的轶事证据支持以下事实mRuby2有时会结合相当紧密到感兴趣的蛋白质，通过多个层析步骤剩余的约束。此行为是明显不希望的，但意外的红宝石络合物通常可以分离并用色谱除去。目前还不清楚这是否是分子伴侣的行为，已经观察到MBP 32。 获得晶体后，有通常面临着COI仍有许多挑战未在此协议解决导致线圈蛋白质。最常见的是衍射质量差，或者是由于不完全成形的晶格或各向异性衍射。还有的地方，以协助各向异性衍射33的工具，而这些一直为解决一些卷曲螺旋结构18,20的关键。许多晶体病状是不幸难以迅速克服，因此它往往是谨慎以搜索具有增强的衍射性质的附加的晶体形式。这是由成千上万的条件机器人筛选促进。可替代地有许多结晶后技术来提高衍射质量如脱水，或播种34。

通过X射线晶体结构测定作出了现代生物学的各个领域基础性的贡献;提供支持生活以及他们如何与在各种情况下彼此相互作用的大分子的原子视图;让我们来了解导致疾病和提供机会来合理设计药物来治疗疾病的机制。晶体早已确定大分子结构的主要实验技术，目前占结构数据库（www.rcsb.org）的89.3％。这种技术有很多优点，包括非常高的分辨率的潜力，以可视化的大分子具有宽范围的尺寸，相对容易收集数据，并以可视化的大分子如何与溶剂以及配体相互作用的机会的能力。 尽管在重组蛋白表达1,2，PUR众多技术改进ification 3，并用于产生这些系统4，在结晶过程中的最大障碍分子生物学仍然生长衍射质量晶体的能力。这一直是尤其如此，它含有大量的卷曲螺旋结构域的蛋白。据估计，所有氨基酸的多达5％的卷曲线圈5,6内发现的，使之成为一个非常常见的结构特征7，但这些蛋白质通常更难以纯化和结晶比球状蛋白8-10 。这是由以下事实卷曲螺旋结构域通常是一个较大的蛋白质的范围内发现，因此正确预测这些结构域的边界是至关重要的，以避免非结构化的或柔性的序列，其通常是有害的结晶的包含进一步加剧。 在这里，我们提出了一个概念框架，结合基于网络的计算与分析experimenta如何选择结构的研究，以及如何准备和结晶前尝试表征蛋白质样品的蛋白质片段（S）:从替补l数据，通过结晶过程的初始阶段，包括帮助指导用户。我们我们的分析集中在含有大量卷曲螺旋域的两种蛋白质，蘑菇时（SHRM）和Rho激酶（岩）。这些蛋白质被选择，因为它们都包含卷曲螺旋域，并且已知以形成生物相关复杂11-16。蘑菇时和Rho激酶（岩）被预测分别含有卷曲螺旋的〜200和680个残基，多个部分，其中已被结构17-20表征。这里所描述的方法提供了一种改进的工作流程，以快速识别含蛋白卷曲螺旋的片段，就可以经得起为结晶，然而，所描述的技术可以很容易地适于大多数蛋白或蛋白复合物或修改，以结合高通量approaCHES为可用。最后，这些方法通常是廉价的，并且可以由用户在几乎所有经验水平来执行。

<table><tbody><tr><td>BL21(DE3) Rosetta</td><td>Emd Millipore</td><td>70954-3</td><td></td></tr><tr><td>BL21(DE3) Star</td><td>ThermoFisher Scientific</td><td>C601003</td><td></td></tr><tr><td>BL21(DE3) Codon Plus</td><td>Agilent Technologies</td><td>230245</td><td></td></tr><tr><td>Lysozyme</td><td>Spectrum Chemical Mfg Corp</td><td>L3008-5GM</td><td></td></tr><tr><td>Ni-NTA resin</td><td>Life Technologies</td><td>25216</td><td></td></tr><tr><td>SubtilisinA</td><td>Spectrum Chemical Mfg Corp</td><td>S1211-10ML</td><td></td></tr><tr><td>24 well Cryschem Plate</td><td>Hampton research</td><td>HR3-160</td><td></td></tr><tr><td>INTELLI-PLATE &amp;nbsp;96:</td><td>Art Robbins Instruments</td><td>102-0001-03</td><td></td></tr><tr><td>PEG 3350</td><td>Hampton research</td><td>HR2-591</td><td></td></tr><tr><td>PEG 8000</td><td>Hampton research</td><td>HR2-515</td><td></td></tr><tr><td>PEG 400</td><td>Hampton research</td><td>HR2-603</td><td></td></tr><tr><td>PEG 4000</td><td>Hampton research</td><td>HR2-605</td><td></td></tr><tr><td>pcDNA3.1-Clover-mRuby2</td><td>Addgene</td><td>49089</td><td></td></tr><tr><td>Overnight Express Autoinduction System 1</td><td>Emd Millipore</td><td>71300</td><td></td></tr><tr><td>Lysogeny Broth powder</td><td>ThermoFisher Scientific</td><td>12795027&amp;nbsp;</td><td></td></tr></tbody></table>

combining wet and dry lab techniques to guide the crystallization of large coiled-coil containing proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

的图描绘在本系统中使用的工作流程示于图1，并包括三个主要阶段。序列的计算分析用于开发关于感兴趣的卷曲螺旋蛋白的结构域边界的假设。所述Shrm2 SD2域的注释分析的一个例子示于图2。在该图中，目标是确定在细胞骨架调节蘑菇时称为SD2的C末端保守结构域可能域边界。从这个分析是生成三个不同的集合假想域边界的包含跨越整个保守SD2或最小片段的C-末端这结束了作为用于结晶的最佳人选邻近卷曲螺旋片段。从该序列分析鉴定候选然后在小规模使用具有mRuby2蛋白质融合（ 图3）FO快速测试ř有效的分析。表示小尺度（50毫升培养的）从两个纯化他10 -mRuby2标记蛋白示于图4。在该图中，不溶性蛋白质的行为相比，其可溶性对应物作为显而易见的。差的表达或不溶性蛋白质融合物容易且快速地以这种方式确定的。通过有限的蛋白水解蛋白片段的生化分析显示于图5，适用于各种使用枯萎红宝石系统片段的描述或与分离和纯化的蘑菇时SD2域。在图5A中，对应于假想域边界鼠标SHRM SD2的两个变体＃2和＃1在图3中采用的蛋白酶浓度的梯度，从（0〜1.0％），在室温下30分钟消化。这两个都有效地降解为在适中的酶浓度的宿主的更小的产品。 图5B示出了在SAMË实验使用的假设边界＃3 果蝇 SHRM SD2执行。这种蛋白质没有结晶，其结构已经描述19。检查Ruby的融合时，按上述从协议产生的蛋白分解分析也可以是有用的。如在图5C中 ，与人类Shrm2 SD2（1427年至1610年）一红宝石融合蛋白所示，用在室温下的非特异性的蛋白酶枯草杆菌的高浓度（0.025％），在指定的时间点进行消化。在这里，Ruby和人力资源管理学会之间的连接立刻被切割正如所预料的。此外，次要产物形成表示该蛋白有两个其它的蛋白酶敏感区域，但是，这是在2分钟内产生的降解产物保持整个实验表明该蛋白质的其余部分基本完好实际上是相当稳定的。 一个补充办法是使用原生PAGE analys所示是在图6中。在图6A中 ，红宝石标记的人SHRM SD2（1627至10年），以及蛋白质含有内SHRM所指示的点突变是通过非变性PAGE进行分析。在这个实验中，野生型蛋白质（其形成结晶）运行为两个不同的频带，而三突变体不结晶具有显着不同的行为。为了证明该系统还可以在蛋白质复合物是有用的，各种蘑菇时摇滚复合物通过在图6B变性PAGE进行分析。在这种情况下，使用人Shrm2 SD2的相同片段（一四二七年至1610年），以帮助澄清分析，而分别利用人类岩不同片段。这种做法表明，使用Rock 700-906和746-906有多个品种，是油污，少同质形成的复合物。利用788-906配合进行了改进，虽然不显着，而这个物种是能够结晶，虽然水晶花了几个星期，形成和反对tained降解蛋白质的岩石。使用Rock 834-913生成络合物形成的凝胶上的单个和更均匀的物种和容易结晶过夜。 图7示出了一组用来通知在结晶试验蛋白质样品的行为共同结晶条件，并可以与任何蛋白质通常使用。理想的情况下，将获得的清晰和沉淀条件的混合。不形成沉淀的蛋白质所需要的高浓度或更严格的缓冲状况，同时那些在许多条件下沉淀应在较低的蛋白质浓度，或与促进蛋白质溶解性缓冲条件下使用。 <img alt="图1" src="/files/ftp_upload/54886/54886fig1.jpg" /> 图1: 工作流程图。广义图描绘计算序列分析和生化和整合其他潮湿的实验室技术转化为划定域边界，并确定蛋白片段结晶的全面战略。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54886/54886fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54886/54886fig2.jpg" /> 图2: 用于从蘑菇时卷曲螺旋SD2域注释序列分析。计算的重叠分析为蘑菇时SD2域，包括由CLUSTAL欧米加生成和内Jalview（步骤1.1）由序列同一性着色的多序列比对。还包括被预测的二级结构的，无序的序列的预测，和蘑菇时SD2域的卷曲螺旋的预测（步骤1.2）。假想域边界（步骤1.3）被表示为是所观察到的边界和二级结构所揭示的后续晶体学分析19的元素。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54886/54886fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54886/54886fig3.jpg" /> 图 3: 他10 -mRuby2表达系统的图。 （ 一 ）示意图表达载体在这项研究中使用他的10 -mRuby2-XH2载体。从该载体多克隆位点（B）的图。蛋白编码序列通常插入通过BamHI和EcoRI克隆位点该载体。所述mRuby2蛋白的极端C-末端以红色示出，TEV蛋白酶切割位点被指示与支架和与显示为红色三角形裂解的位点。的mRuby2和TEV站点之间的连接物序列示于青色。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54886/54886fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54886/54886fig4.jpg" /> 图4: 二mRuby2代表小规模纯化标记的融合蛋白。 （A）的细菌培养物表达的Ruby SHRM SD2或已知不溶性无关红宝石融合蛋白的样品的图像被描绘。没有表达一个Ruby标签的蛋白的单独文化作为对比。 （B）从培养上述进行成像以下裂解和离心以30,000 xg离心30分钟将可溶性级分，表明可溶性红宝石SHRM SD2的可视化。在结合后的的Ni-NTA珠（C）的图像和表示红宝石SHRM SD2被固定在随后的洗涤步骤珠。 （D）在步骤2.3.6和2.3.7中所述的洗涤和洗脱步骤的样品表明，红宝石SHRM SD2融合保持结合到树脂上，而在高达80 mM咪唑的存在下，并且有效地洗脱出来的柱用1M咪唑的洗脱缓冲液。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54886/54886fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54886/54886fig5.jpg" /> 图5:从不同蘑菇时蛋白候选SD2域的有限的蛋白水解。从各种蘑菇时蛋白质4 SD2域比较。 （A和B）指示的人力资源管理协会SD2片段与通过SDS-PAGE分析蛋白酶胰蛋白酶的浓度梯度从0至1.0％，其结果孵育。蛋白质片段机智的关系小时假想边界表示。采用在协议中所述的时间过程的方法他10 -mRuby2，蘑菇时SD2融合蛋白的蛋白水解有限消化（C），SDS-PAGE分析。该反应发生以0.025％在室温下胰蛋白酶。 Ruby和蘑菇时SD2之间的连接体的消化是迅速并且用作内部对照。该共同净化与他的10 -mRuby2，蘑菇时SD2融合蛋白的推测降解产物指示（*）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54886/54886fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54886/54886fig6.jpg" /> 图6:观察使用天然凝胶蛋白行为的变化。 （A）的10％非变性PAGE表明改变mRuby2-Shroo的性质的突变体的效果米SD2。在这些条件下人力资源管理学会SD2运行作为两个独立的频段，而SD2内指定的点突变显示一系列的异常迁徙模式。 （B）蘑菇时SD2-岩复合物的使用不同版本的岩石形成和母语PAGE分析比较。蘑菇时SD2和岩石激酶的指示片段（包括卷曲螺旋蛋白）之间的配合被变性PAGE解决。含有岩788-906多星期，但含有岩834-913复杂的迅速结晶后的复获得晶体。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54886/54886fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54886/54886fig7.jpg" /> 图 7: 结晶快速主屏幕。 （A）共同合作的结晶的快速屏幕nditions是用来评估在结晶试验蛋白质样品的行为。 （ 二 ）简单的评分矩阵的例子，以评估结晶滴。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54886/54886fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins at JoVE.com

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

结合干湿实验室技术指导大型卷曲螺旋含有蛋白质的结晶

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

combining-wet-dry-lab-techniques-to-guide-crystallization-large

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.9K 次观看.  University of Pittsburgh. We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

视频: Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

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 ...

科研

生物化学

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

从构造到晶体 - 迈向β桶外膜蛋白的结构解析

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

化学

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

免疫与感染

High-Throughput Protein Crystallization via Microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature. This protocol describes a straightforward high-throughput (HTP) workflow for screening crystallization conditions through the use of a 96-well dialysis plate. These plates follow the Society for Biomolecular Screening (SBS) standard and can be easily set up using any standard crystallization laboratory. Once optimal conditions are identified, large quantities of crystals (hundreds of microcrystals) can be produced using the dialyzer. To validate the robustness and versatility of this approach, four different proteins were crystallized, including two membrane proteins.

High-Throughput Protein Crystallization via Microdialysis

The presented protocol describes a straightforward approach for screening protein crystallization conditions and crystal growth using a 96-well high-throughput dialysis plate. The use of dialyzer tubes for the large-scale growth of microcrystals is also demonstrated for serial crystallography and MicroED applications.

通过微透析 实现 高通量蛋白质结晶

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug ...

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 ...

生物学

脂质双细胞中 ABC 转运蛋白膜蛋白的结晶

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an aqueous environment is typically achieved by employing detergents. These detergents disintegrate the lipid bilayer and solubilize the proteins. The intrinsic habitat of membrane proteins within the lipid bilayer poses a challenge in maintaining their stability and uniformity in solution for structural characterization. Bicelles, which comprise a blend of long and short-chain phospholipids and detergents, replicate the natural lipid structure. The utilization of lipid bicelles and detergents serves as a suitable model system for obtaining high-quality diffraction crystals, specifically to determine the high-resolution structure of membrane proteins. Through these synthetic microenvironments, membrane proteins preserve their native conformation and functionality, facilitating the formation of three-dimensional crystals. In this approach, the detergent-solubilized heterodimeric ABCG5/G8 was reintegrated into DMPC/CHAPSO bicelles, supplemented with cholesterol. This setup was employed in the vapor diffusion experimental procedure for protein crystallization.

作者聚焦：用于 ABC 转运蛋白转运蛋白的 Bicelle 结晶设置以推进药物开发 

This protocol describes a setup for the crystallization of the sterol transporter ABCG5/G8. ABCG5/G8 is reconstituted into bicelles for hanging-drop crystallization. The protocol does not require specialized materials or substrates, making it accessible and easy to adapt in any laboratory for determining the protein structure through X-ray crystallography.

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an ...

结合干湿实验室技术指导大型卷曲螺旋含有蛋白质的结晶

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分子生物学实验室中大分子结晶的自动化协议

从构造到晶体 - 迈向β桶外膜蛋白的结构解析

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

通过微透析实现高通量蛋白质结晶

X射线晶体学的蛋白质结晶

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

结合干湿实验室技术指导大型卷曲螺旋含有蛋白质的结晶

摘要

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分子生物学实验室中大分子结晶的自动化协议

从构造到晶体 - 迈向β桶外膜蛋白的结构解析

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

通过微透析 实现 高通量蛋白质结晶

X射线晶体学的蛋白质结晶

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

通过微透析实现高通量蛋白质结晶