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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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+interaction+of+ROCKI+with+the+switch+regions+of+RhoA.">Structural insights into the interaction of ROCKI with the switch regions of RhoA.</a> J Biol Chem. 279 (8), 7098-7104 (2004).</li><li>Mohan, S., et al. <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+a+highly+conserved+domain+of+Rock1+required+for+Shroom-mediated+regulation+of+cell+morphology.">Structure of a highly conserved domain of Rock1 required for Shroom-mediated regulation of cell morphology.</a> PLoS One. 8 (12), e81075 (2013).</li><li>Mohan, S., et al. <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+Shroom+domain+2+reveals+a+three-segmented+coiled-coil+required+for+dimerization,+Rock+binding,+and+apical+constriction.">Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction.</a> Mol Biol Cell. 23 (11), 2131-2142 (2012).</li><li>Tu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+coiled-coil+domain+from+human+ROCK+I.">Crystal structure of a coiled-coil domain from human ROCK I.</a> PLoS One. 6 (3), e18080 (2011).</li><li>Sievers, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+scalable+generation+of+high-quality+protein+multiple+sequence+alignments+using+Clustal+Omega.">Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.</a> Mol Syst Biol. 7, 539 (2011).</li><li>Shapiro, A. L., Vinuela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Biophys Res Commun. 28 (5), 815-820 (1967).</li><li>Diezel, W., Kopperschlager, G., Hofmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+procedure+for+protein+staining+in+polyacrylamide+gels+with+a+new+type+of+Coomassie+Brilliant+Blue.">An improved procedure for protein staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue.</a> Anal Biochem. 48 (2), 617-620 (1972).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+limitations+of+clear-native+PAGE.">Advantages and limitations of clear-native PAGE.</a> Proteomics. 5 (17), 4338-4346 (2005).</li><li>Jungbauer, A., Hahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-exchange+chromatography.">Ion-exchange chromatography.</a> Methods Enzymol. 463, 349-371 (2009).</li><li>Yu, C. M., Mun, S., Wang, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+the+effects+of+reversible+dimerization+in+size+exclusion+chromatography.">Theoretical analysis of the effects of reversible dimerization in size exclusion chromatography.</a> J Chromatogr A. 1132 (1-2), 98-108 (2006).</li><li>Giege, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+historical+perspective+on+protein+crystallization+from+1840+to+the+present+day.">A historical perspective on protein crystallization from 1840 to the present day.</a> FEBS J. 280 (24), 6456-6497 (2013).</li><li>Lobley, A., Sadowski, M. I., Jones, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pGenTHREADER+and+pDomTHREADER:+new+methods+for+improved+protein+fold+recognition+and+superfamily+discrimination.">pGenTHREADER and pDomTHREADER: new methods for improved protein fold recognition and superfamily discrimination.</a> Bioinformatics. 25 (14), 1761-1767 (2009).</li><li>Marsden, R. L., McGuffin, L. J., Jones, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+protein+domain+assignment+from+amino+acid+sequence+using+predicted+secondary+structure.">Rapid protein domain assignment from amino acid sequence using predicted secondary structure.</a> Protein Sci. 11 (12), 2814-2824 (2002).</li><li>Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., Sternberg, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Phyre2+web+portal+for+protein+modeling,+prediction+and+analysis.">The Phyre2 web portal for protein modeling, prediction and analysis.</a> Nat Protoc. 10 (6), 845-858 (2015).</li><li>Elsliger, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+JCSG+high-throughput+structural+biology+pipeline.">The JCSG high-throughput structural biology pipeline.</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 66 (Pt 10), 1137-1142 (2010).</li><li>Bach, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+maltose-binding+protein+as+a+molecular+chaperone+for+recombinant+intracellular+cytoplasmic+single-chain+antibodies.">Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies.</a> J Mol Biol. 312 (1), 79-93 (2001).</li><li>Strong, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+structural+genomics+of+complexes:+crystal+structure+of+a+PE/PPE+protein+complex+from+Mycobacterium+tuberculosis.">Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis.</a> Proc Natl Acad Sci U S A. 103 (21), 8060-8065 (2006).</li><li>Heras, B., Martin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-crystallization+treatments+for+improving+diffraction+quality+of+protein+crystals.">Post-crystallization treatments for improving diffraction quality of protein crystals.</a> Acta Crystallogr D Biol Crystallogr. 61 (Pt 9), 1173-1180 (2005).</li></ol>

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。 <p class...

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。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Rosetta</td>
			<td style="width:263px;">Emd Millipore</td>
			<td style="width:136px;">70954-3</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Star</td>
			<td style="width:263px;">ThermoFisher Scientific</td>
			<td style="width:136px;">C601003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Codon Plus</td>
			<td style="width:263px;">Agilent Technologies</td>
			<td style="width:136px;">230245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lysozyme</td>
			<td style="width:263px;">Spectrum Chemical Mfg Corp</td>
			<td style="width:136px;">L3008-5GM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ni-NTA resin</td>
			<td style="width:263px;">Life Technologies</td>
			<td style="width:136px;">25216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">SubtilisinA</td>
			<td style="width:263px;">Spectrum Chemical Mfg Corp</td>
			<td style="width:136px;">S1211-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well Cryschem Plate</td>
			<td>Hampton research</td>
			<td style="width:136px;">HR3-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INTELLI-PLATE &#160;96:</td>
			<td style="width:263px;">Art Robbins Instruments</td>
			<td style="width:136px;">102-0001-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 3350</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 8000</td>
			<td style="width:263px;">Hampton research</td>
			<td style="width:136px;">HR2-515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 400</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 4000</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">pcDNA3.1-Clover-mRuby2</td>
			<td style="width:263px;">Addgene</td>
			<td style="width:136px;">49089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Overnight Express Autoinduction System 1</td>
			<td style="width:263px;">Emd Millipore</td>
			<td>71300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysogeny Broth powder</td>
			<td style="width:263px;">ThermoFisher Scientific</td>
			<td>12795027&#160;</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...

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

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

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

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

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

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

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

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

科研

生物化学

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The analysis does not usually require extensive sample preparation; structural and chemical information can be obtained without labeling. However, each Raman image contains thousands of spectra; this raises difficulties when extracting hidden information, especially for components with similar chemical structures. This work introduces a multivariate analysis to address this issue. The protocol establishes a general method to visualize the main components, including lignin, cellulose, and hemicellulose within the plant cell wall. In this protocol, procedures for sample preparation, spectral acquisition, and data processing are described. It is highly dependent upon operator skill at sample preparation and data analysis. By using this approach, a Raman investigation can be performed by a non-specialist user to acquire high-quality data and meaningful results for plant cell wall analysis.

This protocol aims to present a general method to visualize lignin, cellulose, and hemicellulose in plant cell walls using Raman imaging and multivariate analysis.

结合拉曼成像和多变量分析可视化植物细胞壁中的木质素，纤维素和半纤维素

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

人的 ikkknα的生产、结晶和结构测定指南

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

蛋白质的表征(按尺寸-排除色谱与多角度光散射相结合(SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

使用含有微型基因的Alu元素来分析圆形RNA

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

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

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

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

室温下的片上结晶和大规模连续衍射

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

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

通过荧光体积排阻色谱快速评估膜蛋白质量

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...