We acknowledge financial support for the project from the French grant REPLIPOX ANR-13-BSV8-0014 and by research grants from the Service de Sant&#233; des Arm&#233;es and the D&#233;l&#233;gation G&#233;n&#233;rale pour l'Armement. We are thankful to the ESRF for the SAXS beam time. We thank Andrew McCarthy, Gordon Leonard, Wim Burmeister, and Guy Schoehn for financial and scientific support.

1.离线准备的说明和样本生成一个D5缺失的蛋白质注:D5 323-785构建体（ 图1A）中的克隆，表达，和纯化如所述18。 <ol><li> 克隆构建成pProEx HTb中矢量 <ol><li>执行使用5&#39;-GCGCCATGGGTAATAAACTGTTTAATATTGCAC-3&#39;和5&#39;-ATGCAAGCTTTTACGGAGATGAAATATCCTCTATGA-3&#39;引物和聚合酶的PCR反应混合物，按照制造商的建议上D5R聚合酶链式反应（PCR）。 </li><li>通过离心柱纯化PCR片段，按照制造商的建议。 </li><li>消化的PCR片段并与NcoI和HindIII限制性内切酶的质粒，按照制造商的缓冲组合物和培养时间的建议。 </li><li>运行在0.5×TAE缓冲液在1％琼脂糖凝胶（所述片段的20mM Tris，10mM的醋酸，和0.5mM EDTA）并弄脏用DNA染料的凝胶。 </li><li>揭露凝胶紫外光。 注意:请佩戴个人安全装备！切出的消化的PCR片段的带和消化的质粒和使用凝胶纯化旋转柱试剂盒纯化该DNA，按照制造商的建议。 </li><li>使用快速连接试剂盒，按照制造商的建议，结扎纯化的PCR片段插入质粒。 </li><li>通过热休克从连接反应转化为质粒扩增优化主管细菌转化的产物。 <ol><li>一半连接产物添加到细菌的小瓶。 </li><li>在冰上孵育反应管20分钟。 </li><li>孵育在42℃的管30秒。 </li><li>放置在冰上的管2分钟。 </li><li>添加的Luria BERTANI毫升（LB）肉汤（米勒）不含抗生素1，并让细胞在37℃恢复1小时。 </li><li>降速细胞在16100 XG FO的R 3在台式离心机微管，并除去大部分的介质。 </li><li>传播细胞到具有氨苄青霉素（50微克/毫升终浓度）的LB琼脂平板，并让菌落在37℃生长过夜。 </li></ol></li><li>将一个菌落分为3毫升的LB氨苄青霉素（50微克/毫升最终）和壮大文化在37℃，在160 rpm振摇过夜。 </li><li>按照小量制备试剂盒提取质粒的指令。 </li><li>发送质粒进行测序的样品，并分析了没有突变和为正确的插入序列。 </li><li>变换pProEx HTb中D5 323-785构建成表达优化菌（使用1μL，并按照步骤1.1.7步骤）。传播细菌到具有氨苄青霉素（50微克/毫升终浓度）的LB琼脂板上。 </li><li>要创建开始培养，把一个菌落在300ml的LB氨苄青霉素（50微克/毫升终浓度）并在37℃下生长的细菌，在160ř摇动下午过夜。 </li></ol></li><li>接种12×1升的LB氨苄青霉素的存在下（50微克/毫升终浓度），每20毫升的pProEx HTb中D5 323-785细菌起子培养的，在37℃，直到OD 600 = 0.3为止。降低温度至18℃，并在的OD 600 = 0.5，用1mM IPTG诱导表达。孵育文化，在160转，18℃过夜摇晃他们。 </li><li>收获离心细菌以50,000 xg离心并在4℃下进行30分钟。重悬细菌粒料在约150毫升的裂解缓冲液（50毫摩尔Tris-盐酸[pH为7]，150 mM氯化钠，5mM的MgCl 2的，10毫β巯基乙醇，和10％甘油）用1×蛋白酶抑制剂和25个单位（ U）/ 10毫升的DNA酶。通过裂解超声冰上细菌（1英寸探头，50％的功率，0.5的脉搏，0.5秒暂停，3个5分钟）。 </li><li>加载上清液到镍亲和柱（Ni柱，1.5毫升床体积），以及用b的10个柱体积（CV）的洗inding缓冲液（50mM的Tris-HCl [pH为7]，150毫摩尔NaCl，10毫β巯基乙醇，和10％甘油），洗涤液10的CV（50毫摩尔Tris-盐酸[pH为7]，1 M氯化钠，10毫β巯基乙醇，和10％甘油），和咪唑洗10 CV（50毫摩尔Tris-盐酸[pH为7]，150毫摩尔NaCl，10毫β巯基乙醇，10％甘油，和30 mM咪唑）。洗脱D5 323-785（20毫Tris-盐酸[pH为7]，150毫摩尔NaCl，10毫β巯基乙醇，10％甘油，和200mM的咪唑）。 </li><li>检查通过十二烷基硫酸钠（SDS） - 聚丙烯酰胺凝胶电泳（PAGE）在不同纯化步骤。 <ol><li>负载2-20μL的各纯化步骤的流通用1×上样染料混合（2次:4％SDS，20％甘油，120毫摩尔Tris-盐酸[pH为6.8]，和0.02％重量/体积的溴酚蓝）上的10 ％SDS凝胶和在1倍的Laemmli 200伏运行它们运行缓冲液（25mM的Tris，0.192甘氨酸和0.1％SDS）中。 </li><li>染色用现成的使用考马斯蓝溶液的凝胶。 </li></ol></li><li> EXC焊割使用根据制造商的手册脱盐柱结合缓冲液洗脱的蛋白的缓冲器。 </li><li>通过加入烟草蚀纹病毒核包涵体一个内肽酶 （TEV，1毫克/ 100毫克蛋白）的蛋白质溶液裂解His-标签。在室温下孵育过夜。 </li><li>通过执行SDS-PAGE检查消化（见步骤1.5） </li><li>平衡在结合缓冲液中Ni柱。让蛋白质溶液通过用2的CV咪唑洗涤缓冲液洗珠而流过收集。 </li><li>用离心浓缩至0.5毫升的最终体积浓缩蛋白质。 </li><li>注入浓缩的蛋白质上与凝胶过滤缓冲液平衡的大小排阻柱（20毫Tris-盐酸[pH为7]，150毫摩尔NaCl，10％甘油和1mM二硫苏糖醇[DTT]）。 </li><li>通过流入0.5毫升馏分收集。 </li><li>检查峰样品通过在蛋白质的存在和纯度进行SDS-PAGE（见步骤1.5）。 </li><li>结合D5 323-785的洗脱峰的级分。稀释样品至25毫摩尔NaCl和5％的甘油，同时保持Tris和DTT浓度恒定（5毫升蛋白质溶液在20mM的Tris-HCl [pH为7]，150毫摩尔NaCl，10％甘油，和1mM DTT，首先添加5毫升的20mM的Tris-HCl [pH为7]和1mM DTT的，然后添加20毫摩尔Tris-盐酸[pH为7]，5％甘油，和1mM DTT）的20毫升。 </li><li>加载样品上的离子交换柱。使用缓冲液A（20毫摩尔Tris [pH为7，25 mM氯化钠，5％甘油，和1mM DTT）和缓冲液B（20毫摩尔Tris [pH为7]，1 M氯化钠，5％甘油，和1mM DTT）以形成的盐梯度从25mM至1M。 </li><li>收集洗脱的蛋白在1.5毫升的级分。 </li><li>通过进行SDS-PAGE检查峰样品中蛋白质的存在和纯度（参见步骤1.5和图1B） </li><li> Reconcentrate在离心浓缩至0.5毫升的最终体积的蛋白质溶液。 </li><li> ReruN于在凝胶过滤缓冲液中的大小排阻柱浓缩的蛋白质（的20mM Tris - 盐酸[pH为7]，150 mM氯化钠，5％甘油，和1mM DTT;见步骤1.11）。 </li></ol> 2，数据收集注:尽早申请束的时间。对于ESRF，可用的接入类型以及如何提交申请指南可以在这里找到: <a href="http://www.esrf.eu/UsersAndScience/UserGuide/Applying">http://www.esrf.eu/UsersAndScience/UserGuide/Applying</a> 。接受该建议，并为实验的邀请后，所有参与者都完成安全培训。培训的有效性后，填写"A型"（通过用户ESRF门户）宣布，研究人员访问了实验束线，对样品所需的安全信息。联系当地联络人讨论实验。 <ol><li> SEC-SAXS数据收集 注:对于ONLINE纯化，使用高效液相色谱（HPLC）安装在BM29系统。该系统包括一个在线脱气，二元泵，用于缓冲器选择和梯度，自动取样器，一个UV-VIS阵列photospectrometer，和一个电导阀的。它被直接连接到SAXS曝光单元19的流过毛细管。 <ol><li>把1毫升的凝胶过滤缓冲的一边。连接缓冲液瓶向SEC系统（默认端口号为A）。 </li><li>选择取决于所使用的柱的流速。注意:对于这里使用的尺寸排阻柱，流速为0.1毫升/分钟。定义列的最大压力，注意回压，以及采集时间设置为至少1.2 CV。 </li><li>通过"自动清理"功能，冲洗泵和上游管。 </li><li>等到系统充满了缓冲和连接柱。通过使至少1.5 CV平衡色谱柱。 </li><li>连锁双雄和测量缓冲步骤2.1.1作废，使用自动进样器的信号变化，以控制由于辐射伤害。仅当有到缓冲无辐射损伤继续。 </li><li>束线控制系统切换到HPLC模式。做缓冲的试运行，采集200帧，每帧1秒。写在求和强度情节由检测给定的计数数目。 注意:信号应与先前测量的缓冲器，并随着时间的推移的求和强度应保持不变。如果信号不匹配或不恒定，需要系统的较长的平衡。 </li><li>降速样品以13,000rpm XG在台式微型管离心至少10分钟。 </li><li>填写样品到HPLC兼容的玻璃小瓶（提供），放入自动注射器。注意不错。 </li><li>互锁使用标准程序实验双雄，如用户安全培训解释。 </li><li>登录到并通过束线控制软件创建的数据存储的文件夹。 </li><li>程序中使用了"快批"功能的高效液相色谱系统。设置的存储位置的UV数据在步骤2.1.10创建文件夹。确保激活UV数据的自动ASCII转换，存储ASCII文件在同一文件夹。 <ol><li>选择注射量和井位。添加测量通过按下"开始"按钮来排队。该软件会要求保存批处理。准备储蓄，但不要按"保存"按钮，这将自动启动测量。 </li></ol></li><li>设立在光束线控制软件数据收集参数。选择的帧的数量，以使总的数据采集时间是在稍过量的在步骤2.1.2中定义的采集时间。 </li><li>打开安全快门，并使用束线控制软件开始SAXS数据收集。验证数据是心病rectly收购。新收集的数据进行比较，以在步骤2.1.6收集到的数据，并检查了至少100帧在总结强度计数的数量保持不变，值从步骤2.1.6匹配。 <ol><li>启动SEC按"保存"按钮运行，如步骤2.1.11准备，并注意在注射完成和UV数据采集开始在camserver软件中显示的帧数。 </li></ol></li><li>数据收集后，打开ISPyB数据库20在打开"数据采集"选项卡，点击"开始"按钮，进入数据集和自动分析21的结果。 </li></ol></li><li> IEC-SAXS数据收集 注:代替在IEC实验通常所施加的连续的线性梯度，使用逐步梯度，其中缓冲液B的量在预先设定的离散的步骤增加。 <ol><li>为SAMPL创建HPLC程序电子无缓冲运行。编程分步梯度从0％缓冲液B开始并达到的缓冲液B 100％，直到经过两CV由5个百分点。 </li><li>把一些每个缓冲区的一边。瓶子将随低盐缓冲液A端口A和一个具有高盐缓冲液B端口B. </li><li>选择的流速和保持低于塔的压力极限（在这个例子中，1毫升/分钟）。 </li><li>如在步骤2.1.3，通过使用"自动清洁"功能冲洗泵和上游管。 </li><li>平衡在低盐缓冲液A的系统（见步骤1.15），并连接所述离子交换柱。等到柱子完全平衡（至少1.5 CV）。 </li><li>使用缓冲区预留步骤2.2.2检查缓冲区A和B使用样品转换，如步骤2.1.5辐射伤害。 </li><li>检查缓冲器出来的列的，如在步骤2.1.6。比较信号记录在步骤2.2.6缓冲区中的信号。注意再次在求和强度情节计数数目。 </li><li>创建的数据存储在缓冲器运行的文件夹。 </li><li>建立数据收集在HPLC系统模式。选择的帧的数量，以使总的数据采集时间是在过量的IEC运行的总时间（参见步骤2.2.1）。 </li><li>打开安全快门，并启动SAXS数据收集。验证它是否正确地进行并仔细检查了总结强度保持在步骤2.2.7指出，至少100帧数值不变。 </li><li>使用HPLC软件的"单次运行"的功能，并开始在步骤2.2.1编程的分步梯度。为注射剂，为了在这个步骤中注入没有样品设置瓶号为-1。写下在程序启动时获得的帧数。 </li><li>创建示例运行的HPLC程序。编程分步梯度从缓冲液B估计的0％缓冲液B中的每个峰在步骤1.1.5脱机测量的百分比开始（ <str翁&gt;图1B）。置正好1个点以下和感兴趣的峰高于1.5分的至少3个步骤，每2.5 CV（ 图1C）。加在100％缓冲液B的最终步骤为2.5的简历。 </li><li>降速样品以13,000rpm XG在台式微型管离心至少10分钟。 </li><li>通过用20mM的Tris-HCl [pH为7]，10％甘油，和1mM DTT混合，稀释D5 323-785成缓冲液A（25毫米）的盐浓度。 </li><li>手动加载样品到柱上。把缓冲区中的输入管插入稀释的样品暂停泵避免气泡产生后形成的。断开检测器的列直接收集从柱中流通。 <ol><li>当样本容器几乎是空的，则返回该输入管成缓冲液A（再次暂停泵而移动所述管），并重新启动用缓冲液A泵送至样品从管转移到柱上。一旦整个样本已经加载，通过2 CV缓冲液A，然后重新连接列探测器。 </li></ol></li><li>对于样品运行，创建用于数据存储的文件夹。 </li><li>打开安全快门，并启动SAXS数据收集。验证数据收集正确进行并仔细检查了总结强度保持在步骤2.2.7指出，至少100帧数值不变。 </li><li>用HPLC软件的"单次运行"功能启动步骤2.2.12编程的逐步梯度。为注射剂，为了在这个步骤中注入没有样品设置瓶号为-1。写下在程序启动时获得的帧数。 </li><li>在ISPyB 20打开"数据采集"，按"走出去"看数据集和自动分析21。 </li><li>通过"后运行分析"软件出口从HPLC系统，以ASCII格式的UV数据。 </li></ol></li></ol> 3。SEC-SAXS数据缩减和分析<ol><li>按"转到"按钮打开ISPyB数据采集的数据。确定在该帧的SAXS数据集合的概述对应洗脱峰。验证回转半径在整个峰稳定。 </li><li>确认自动缓冲区修正正确执行。从峰值的中心部分成与实验SAXS数据执行操作文件22和平均他们一个程序装入帧。然后，装入自动生成缓冲文件，并检查是否平均帧的高Q值区和缓冲区帧匹配。 </li><li>整个比较定量使用CORMAP测试23峰采集的帧。 <ol><li>加载覆盖的感兴趣的区域的帧的自动创建减法（* _sub.dat文件）。 </li><li>按"比例"按钮将其扩展到对方。 </li><li> COMPARË他们使用"数据比较"功能。应在整个峰的帧之间没有系统的变化。 </li></ol></li><li>打开所有感兴趣的* -_ sub.dat帧，使用"合并"按钮将它们合并，并保存.dat文件合并文件。 </li><li>确定与在程序中"回转半径"工具的回转半径，并指出在吉尼耶范围中的第一个数据点在低分辨率数据的后面裁剪。 </li><li>确定与在节目中的"距离分布"工具对距离分布函数和保存生成的.out文件为gnom.out。 </li></ol> 4. 从头建模<ol><li>在Unix机器上，运行无对称性限制40× 从头模型重建计划。创建一个新的文件夹和gnom.out文件复制到它。运行程序如下: 为（（i = 1; I &lt;= 40;我++））;做dammif gnom.out -ms -p dammifp1_ $ I; ð一</li><li>类似地，与运行在第二个文件夹六倍对称性对称性限制的从头模型重建方案: 为（（i = 1; I &lt;= 40;我++））;做dammif gnom.out -ms -s P6 -p dammifp6_ $ I; DONE </li><li>对齐所有从头模型重建程序输出文件（* -1.pdb）。在从步骤4.1和4.2两个文件夹，运行damaver -a * -1.pdb。 </li><li>打开所有车型（damaver.pdb），以及在合适的分子可视化软件26过滤，以正确的体积（damfilt.pdb）模型的工会并进行比较。 </li><li>打开一个文本编辑器中damsel.log文件。列为"参考"的文件的底部的模型是最有代表性的模式。 </li></ol> 5. IEC-SAXS数据缩减，分析和建模<ol><li>在该实验的SAXS数据文件22进行操作的程序，加载约50 SAXS从各混合步骤帧缓冲运行，按"平均"按钮，保存结果。 </li><li>基于经由ISPyB可用的自动处理结果，确定实验的哪些帧对应于蛋白质的洗脱，并验证回转（初步）半径在整个峰稳定。 </li><li>加载帧入实验SAXS数据文件处理程序22和平均它们，作为缓冲帧（见步骤5.1）来完成。然后，装载在步骤5.1中产生的缓冲文件并比较（以上4.2纳米-1）高Q的区域，发现从峰，平均匹配的缓冲器。 注:不应该有系统从峰值和缓冲的平均之间的偏移。如果样品曲线似乎两个缓冲曲线之间下降，通过计算平均插值它们的曲线。 </li><li>减去确定为从峰值部分的平均散射曲线的最佳匹配缓冲区，并保存生成的subtr行动文件。另外，使用来自前面和后面的混合步骤的平均缓冲器曲线来估计减法的误差消减曲线。 </li><li>为峰的左和右两半重复步骤5.3和5.4分别并比较的结果，那些来自步骤5.4以检查整个峰的信号的稳定性。 </li><li>按照步骤，从步骤5.4所有三个曲线减去3.5和3.6。 </li><li>比较所有三个减去曲线的结果。 注意:只有减法的误差范围内保持强劲结果是可信的。 </li><li>在步骤在步骤5.3确定最佳的减4.1和4.2进行建模。 </li><li>按照步骤4.3到4.5对齐模式，并确定最有代表性之一。 </li></ol> 6.结果比较<ol><li>运行一个程序叠加的三维结构在美国证券交易委员会的代表机型12（步骤4.5）的ðIEC-SAXS数据（步骤5.9）与P6对称性:supcomb model_sec.pdb model_iec.pdb </li><li>导入model_sec.pdb和model_iec-r.pdb成分子可视化软件。 </li><li>打开modelsec.pdb的.fir文件，并在电子表格中modeliec-r.pdb，创造实验和计算的散射曲线的2D图形。 </li></ol>

<ol>
	<li>Graewert, M. A., Svergun, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+and+progress+in+small+and+wide+angle+X-ray+scattering+(SAXS+and+WAXS).">Impact and progress in small and wide angle X-ray scattering (SAXS and WAXS).</a> Curr. Opin. Struct. Biol. 23, 748-754 (2013).</li><li>Jacques, D. A., Trewhella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-angle+scattering+for+structural+biology-Expanding+the+frontier+while+avoiding+the+pitfalls.">Small-angle scattering for structural biology-Expanding the frontier while avoiding the pitfalls.</a> Protein Sci. 19, 642-657 (2010).</li><li>Kikhney, A. G., Svergun, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+small+angle+X-ray+scattering+(SAXS)+of+flexible+and+intrinsically+disordered+proteins.">A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins.</a> FEBS Lett. 589, 2570-2577 (2015).</li><li>Putnam, C. D., Hammel, M., Hura, G. L., Tainer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+solution+scattering+(SAXS)+combined+with+crystallography+and+computation:+defining+accurate+macromolecular+structures,+conformations+and+assemblies+in+solution.">X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution.</a> Q. Rev. Biophys. 40, 191-285 (2007).</li><li>Vestergaard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+biostructural+changes,+dynamics,+and+interactions+-+Small-angle+X-ray+scattering+to+the+rescue.">Analysis of biostructural changes, dynamics, and interactions - Small-angle X-ray scattering to the rescue.</a> Arch. Biochem. Biophys. , (2016).</li><li>David, G., P&#233;rez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+sampler+robot+and+high-performance+liquid+chromatography:+a+fully+automated+system+for+biological+small-angle+X-ray+scattering+experiments+at+the+Synchrotron+SOLEIL+SWING+beamline.">Combined sampler robot and high-performance liquid chromatography: a fully automated system for biological small-angle X-ray scattering experiments at the Synchrotron SOLEIL SWING beamline.</a> J. Appl. Crystallogr. 42, 892-900 (2009).</li><li>Graewert, 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=Automated+Pipeline+for+Purification,+Biophysical+and+X-Ray+Analysis+of+Biomacromolecular+Solutions.">Automated Pipeline for Purification, Biophysical and X-Ray Analysis of Biomacromolecular Solutions.</a> Sci. Rep. 5, (2015).</li><li>Lambright, 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=Complementary+techniques+enhance+the+quality+and+scope+of+information+obtained+from+SAXS.">Complementary techniques enhance the quality and scope of information obtained from SAXS.</a> ACA Trans. , 1-12 (2013).</li><li>Mathew, E., Mirza, A., Menhart, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid-chromatography-coupled+SAXS+for+accurate+sizing+of+aggregating+proteins.">Liquid-chromatography-coupled SAXS for accurate sizing of aggregating proteins.</a> J. Synchrotron Radiat. 11, 314-318 (2004).</li><li>Round, 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=Determination+of+the+GH3.+12+protein+conformation+through+HPLC-integrated+SAXS+measurements+combined+with+X-ray+crystallography.">Determination of the GH3. 12 protein conformation through HPLC-integrated SAXS measurements combined with X-ray crystallography.</a> Acta Crystallogr. Sect. D. Biol. Crystallogr. 69, 2072-2080 (2013).</li><li>Watanabe, Y., Inoko, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-exclusion+chromatography+combined+with+small-angle+X-ray+scattering+optics.">Size-exclusion chromatography combined with small-angle X-ray scattering optics.</a> J. Chromatogr. 1216, 7461-7465 (2009).</li><li>Hutin, S., Brennich, M. E., Maillot, B., Round, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Online+ion-exchange+chromatography+for+small+angle+X-ray+scattering.">Online ion-exchange chromatography for small angle X-ray scattering.</a> Acta Cryst. , (2016).</li><li>Selkirk, C. <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> Protein Purification Protocols. , 125-131 (2004).</li><li>Yigzaw, Y., Hinckley, P., Hewig, A., Vedantham, G. <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+of+proteins+and+clearance+of+aggregates.">Ion exchange chromatography of proteins and clearance of aggregates.</a> Curr. Pharm. Biotechnol. 10, 421-426 (2009).</li><li>Pernot, P., 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=Upgraded+ESRF+BM29+beamline+for+SAXS+on+macromolecules+in+solution.">Upgraded ESRF BM29 beamline for SAXS on macromolecules in solution.</a> J. Synchrotron Radiat. 20, 660-664 (2013).</li><li>Iyer, L. M., Koonin, E. V., Leipe, D. D., Aravind, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+evolution+of+the+archaeo-eukaryotic+primase+superfamily+and+related+palm-domain+proteins:+structural+insights+and+new+members.">Origin and evolution of the archaeo-eukaryotic primase superfamily and related palm-domain proteins: structural insights and new members.</a> Nucleic Acids Res. 33, 3875-3896 (2005).</li><li>Singleton, M. R., Dillingham, M. S., Wigley, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+mechanism+of+helicases+and+nucleic+acid+translocases.">Structure and mechanism of helicases and nucleic acid translocases.</a> Annu. Rev. Biochem. 76, 23-50 (2007).</li><li>Hutin, 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=Domain+organization+of+vaccinia+virus+helicase-primase+D5.">Domain organization of vaccinia virus helicase-primase D5.</a> J. Virol. , 4604-4613 (2016).</li><li>Round, 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=BioSAXS+Sample+Changer:+a+robotic+sample+changer+for+rapid+and+reliable+high-throughput+X-ray+solution+scattering+experiments.">BioSAXS Sample Changer: a robotic sample changer for rapid and reliable high-throughput X-ray solution scattering experiments.</a> Acta Crystallogr. Sect. D. Biol. Crystallogr. 71, 67-75 (2015).</li><li>De Maria Antolinos, 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=ISPyB+for+BioSAXS,+the+gateway+to+user+autonomy+in+solution+scattering+experiments.">ISPyB for BioSAXS, the gateway to user autonomy in solution scattering experiments.</a> Acta Crystallographica Section D. 71, 76-85 (2015).</li><li>Brennich, M. E., 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=Online+data+analysis+at+the+ESRF+bioSAXS+beamline,+BM29.">Online data analysis at the ESRF bioSAXS beamline, BM29.</a> J. Appl. Crystallogr. 49, (2016).</li><li>Petoukhov, M. V., Konarev, P. V., Kikhney, A. G., Svergun, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATSAS+2.1-towards+automated+and+web-supported+small-angle+scattering+data+analysis.">ATSAS 2.1-towards automated and web-supported small-angle scattering data analysis.</a> J. Appl. Crystallogr. 40, 223-228 (2007).</li><li>Franke, D., Jeffries, C. M., Svergun, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+Map,+a+goodness-of-fit+test+for+one-dimensional+X-ray+scattering+spectra.">Correlation Map, a goodness-of-fit test for one-dimensional X-ray scattering spectra.</a> Nat. Methods. 12, 419-422 (2015).</li><li>Schrodinger, LLC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The PyMOL Molecular Graphics System, Version 1.8. , (2015).</li><li>Jensen, M. 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=Time-resolved+SAXS+measurements+facilitated+by+online+HPLC+buffer+exchange.">Time-resolved SAXS measurements facilitated by online HPLC buffer exchange.</a> J. Synchrotron Radiat. 17, 769-773 (2010).</li><li>Hynson, R. M., Duff, A. P., Kirby, N., Mudie, S., Lee, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+ultracentrifugation+coupled+to+small-angle+X-ray+scattering+on+macromolecular+complexes.">Differential ultracentrifugation coupled to small-angle X-ray scattering on macromolecular complexes.</a> J. Appl. Crystallogr. 48, 769-775 (2015).</li></ol>

The authors have nothing to disclose.

对于许多大分子，用色谱最后纯化步骤之前SAXS数据收集，以获得高质量的数据集需要好。然而，并非所有的样品保持稳定;他们可能倾向于聚集或重新平衡到低聚状态的混合物。因此，在束线的终在线纯化步骤是必需的，以尽量减少为了获得质量最好的SAXS数据纯化和数据采集之间的时间。取决于目的蛋白质的生物物理特性，SEC-SAXS或IEC-SAXS可能被选择以获得最佳的样品质量。这里，对从解旋酶/引物酶D5衍生的蛋白质构建体，这两种技术的解释和讨论。 SEC-SAXS数据的采集变得越来越规范，并提供许多BioSAXS光束线。数据分析，尤其是背景扣除，是相对简单和容易。然而，稳定的缓冲信号和大分子物种的足够的分离仍是至关重要的。因此，关键是要保留足够的时间以彻底平衡色谱柱。这种方法的失败可能是由于所关注，低浓度，和对辐射敏感的缓冲器的蛋白大小相似的持久性污染物。 在实践中，最初，SEC-SAXS很可能被用作首选大多数大分子的样品的方式。尽管如此，许多纯化协议需要由于污染物或聚集的存在下，现有的IEC步骤。鉴于各浓缩和色谱分离步骤是用样品（估计为30-50％）和时间的损失相关联，直接IEC-SAXS是有利的。对于无法通过SEC纯化的样品，它是由于类似尺寸"污染物"的存在，或者因为它们在必要的浓度严重骨料，IEC-SAXS将始终是更好的适合的方法。此外，较高的流速支持由许多IEC列可以帮助减少纯化和测量之间的通行时间。在这里介绍的实施例，IEC用逐步洗脱，其允许D5 323-785的接近的峰从污染物通过仔细选择盐浓度步骤分离使用。原则上，步骤的数目是无限的，但实际上，需要至少1步骤每峰，并没有太多应该选择。对于上述背景减法方法中，关键的是要测量以便找到匹配一个相对高的数目不同的缓冲组合物。 这两项技术共享的缺点是缺乏准确的蛋白质浓度信息。由于这个原因，基于前向散射精确质量测定是不可能的。为球状蛋白质如D5 323-785，所述Porod体积提供了一种替代方案中，尽管不那么精确，质量的估计，但对于高度灵活的或无序的蛋白，这种方法是无效的。 这里提出的逐步梯度IEC-SAXS方法的变化是使用，而不是一个线性渐变。虽然可以根据需要使用分步洗脱来分离次峰与尽可能多的步骤，工作，在线性梯度方法中，它需要仔细优化梯度条件以便分离峰完全启动SAXS之前实验。在这种方法的背景减除可以做到帧方式，可以由各个帧的比较来验证，但它需要更先进的数据处理，和一个专用软件尚不存在。 合适的列的选择对于这两种技术的关键，因为它决定的大分子物种的分离。大小排阻柱中装载能力，可分离大分子的尺寸范围和分辨率的不同，而离子交换柱中的种类和密度变化他们的固定费用。 而这里提出的协议是专用于ESRF束线BM29，适应于任何其他SAXS束线是，原则上，直接的。主要的要求是足够高的X射线通量和一个合适的检测器（最好是单光子计数），在几秒钟或更小的范围内，以获得合理的信号 - 噪声数据，以及在线液相色谱系统能够生成的信息梯度。确切的实施将当然取决于当地束线环境。 使用这两种方法上D5 323-785得到的结果稍有不同。回转半径为比在SEC数据的IEC数据略小，与散射曲线的局部最小值被移位到稍大散射矢量。这意味着，与IEC-SAXS测量的D5 323-785比用SEC-SAXS测量的D5 323-785稍微更紧凑。这可能bÈ由于在样品制备（IEC是更快）的差异，在纯化和测量之间的时间，或不太可能，向SEC纯化的样品中的污染物。有两种方法获得完全独立的珠车型相媲美（ 图1H）。 D5 323-785显示预期中空，六聚体结构18。 总之，在线离子交换和在线大小排阻层析是可直接联接到SAXS 6,7,9-12,25,26重要的生物化学纯化方法。 IEC-SAXS数据的背景减除稍微比SEC-SAXS更加困难和不明确的，但它仍然是可能的。取决于目的蛋白质的生物物理特性，既SEC和IEC-SAXS允许与固有的优点物种分离的优化。提供了该验证步骤（如所描述）被正确地观察到，得到的数据可以被分析机智^ h的信心和模型可以用在社区内提供的标准工具来确定。一起，这两种技术允许范围广泛的生物大分子的在线分离，得到通过标准静态测量不可访问的数据。

BioSAXS是一种强大的工具，以确定纳米尺寸的物体1-4的形状。 X射线的含有大分子，在纳米范围尺寸的溶液的散射，被记录在非常低的角度。这个角度范围包含全局参数信息:回转半径;最大的颗粒内的距离;粒子形状;和折叠，变性，或紊乱的程度。该技术不需要晶体，并且大分子保留在溶液中，因此可以保持在模仿细胞的某些重要参数，如离子强度，pH 等 ，这些因素的知识条件可能有助于确定，例如，目的蛋白质的或生理学相关的寡聚状态，以验证一个复杂的提出的模型。蛋白质 - 蛋白质相互作用的在不同缓冲液条件的表征，创立缺失结构域的模型，同源性模型的细化，并解可以快速并容易地5进行离散折叠和展开状态的终止。 对于任何技术，BioSAXS有其内在的弱点:聚集或变性样品，颗粒的混合物，异构样品，辐射损伤和缓冲的不匹配可能会导致未解释的数据。对于许多分析方法，它是隐式假设样品是单分散，即常常难以得到在实践中的一个要求。在许多情况下，样品的降解是微妙的，不能在其自己的数据被检测到，任何试图解释数据给出不准确或甚至误导的结果。为了克服这些障碍，大小排阻层析（SEC）和SAXS的组合物在许多束线执行，以确保数据的质量和使该技术对于越来越难以样品6-11更容易获得。最近，我们通过开发在线离子交换加入到剧目的新方法色谱法（IEC）的偶联的SAXS 12。这两种技术是开放SAXS了广泛的生物粒子以前不可能分析的。的要使用的方法的选择取决于所关注的粒子的生物物理特性。 SEC可以通过大小，由此需要用于分离至少在表观分子质量10％的差异分离大分子。柱和样品的生理特性的物理限制，例如疏水表面，灵活性和缺乏稳定性，也数据收集，分析和解释变得复杂。 离子交换色谱法，该分离基于它们的电荷的分子，因此，它们的结合亲和力于IEC柱，可被用来代替或附加于SEC。总电荷可通过改变pH值，或改变所述缓冲器的盐浓度，提供了一个相对简单的方法用于受控埃尔容易地操纵从IEC柱分子ution。通过使用电荷，相似类型和分子的大小，这本来是难以分离的分离，可常规符合IEC执行的。此外，IEC具有能够应对稀释的样品，使一个以避免浓缩步骤，其携带变性蛋白质的潜在风险的优点。不幸的是，作为电荷分布是高度样品依赖性，IEC需要关于pH和盐浓度13,14优化。 对于许多蛋白质是难以表达，纯化，或两者，仅低量的样品提供给学习。是有效的，并尽量减少的纯化步骤的数目，因此，损失是很重要的。出于这个原因，最后的纯化步骤是在线之前直接SAXS数据采集，以便提高收集好数据集的可能性。 这里，我们提出和比较网上SEC-SAXS和IEC-SAXS。这两种技术分别对BioSAXS光束线BM29在法国格勒诺布尔15欧洲同步辐射装置（ESRF）来实现。作为试验的情况下，我们使用了牛痘病毒蛋白D5中，这是相当困难的分析结构使用其他方法的D5N和解螺旋酶域。牛痘病毒是痘病毒科家族的成员，并且是98％相同的天花病毒，天花的原因。使用牛痘系统中，我们研究了复制机制，在这里专注于重要的解旋酶 - 引发酶D5。 D5是一个95-kDa蛋白具有N-末端archeo真核引发酶（AEP）域16接着半胱氨酸簇区（水库240-345）16。进一步朝向C末端配备一个D5N域（水库340到460），其总是与D5型解旋酶相关联，并且最后一个超家族3（SF3）解螺旋酶域（水库460-785）17（FIGURË1A）。 D5的解螺旋酶域构建所需要对紧结合DNA中的六聚体环状结构。由于最近SAXS和EM研究中，引发酶的低分辨率结构和旋酶结构域现在已知18。 在这里，我们将展示如何使用网上实现的色谱技术在BioSAXS光束线BM29在ESRF来深入了解C端片段D5的（残留323-785）的结构。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4 dithiothreitol [DTT]</td>
			<td>Euromedex</td>
			<td>EU0006-B</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Mini-PROTEAN TGX Precast Protein Gel</td>
			<td>Biorad</td>
			<td>4561033</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Phusion Flash PCR Master Mix</td>
			<td>ThermoFisher scientific</td>
			<td>F 548</td>
			<td></td>
		</tr>
		<tr>
			<td>acetic acid glacial</td>
			<td>VWR Chemicals (BDH Prolabo)</td>
			<td>20104.298</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose D-5</td>
			<td>Euromedex</td>
			<td>LF45130653</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra -15 Centrifugal filters Ultracel -30K</td>
			<td>Millipore Ltd</td>
			<td>UFC903024</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Euromedex</td>
			<td>EU0400-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Novagene</td>
			<td>70750-3</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue&#160;</td>
			<td>MERCK</td>
			<td>8122</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete protease inhibitor cocktail&#160;</td>
			<td>Roche</td>
			<td>11231400</td>
			<td></td>
		</tr>
		<tr>
			<td>Econo-Pac 10 DG Desalting column</td>
			<td>Bio-rad</td>
			<td>732-2010</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E-5134</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR Chemicals (BDH Prolabo)</td>
			<td>24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Euromedex</td>
			<td>26-128-6405-C</td>
			<td></td>
		</tr>
		<tr>
			<td>HindIII</td>
			<td>Roche</td>
			<td>656313</td>
			<td></td>
		</tr>
		<tr>
			<td>HisSelect HF Nickel Affinity Gel</td>
			<td>Sigma</td>
			<td>H0537</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>VWR Chemicals (BDH Prolabo)</td>
			<td>20252.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>AppliChem Panreac</td>
			<td>A1073,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>InstantBlue</td>
			<td>Expedeon</td>
			<td>ISB1L</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth Miller</td>
			<td>Fluka Analytical</td>
			<td>L3152</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>ICN Biomedicals Inc.</td>
			<td>191421</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR Chemicals (BDH Prolabo)</td>
			<td>27808.297</td>
			<td></td>
		</tr>
		<tr>
			<td>NcoI</td>
			<td>Fermentas</td>
			<td>ER0571</td>
			<td></td>
		</tr>
		<tr>
			<td>One Shot BL21 Star (DE3)&#160;</td>
			<td>ThermoFisher scientific</td>
			<td>C6010-03</td>
			<td></td>
		</tr>
		<tr>
			<td>pProEx HTb vector&#160;</td>
			<td>Addgene</td>
			<td>10711018</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer</td>
			<td>&#160;Eurofins mwg operon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep kit</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel extraction kit</td>
			<td>QIAGEN</td>
			<td>28706</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kid</td>
			<td>QIAGEN</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS&#160;</td>
			<td>Sigma</td>
			<td>75746</td>
			<td></td>
		</tr>
		<tr>
			<td>Superose 6 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>17-5172-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe DNA gel stain</td>
			<td>Invitrogen life technology</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>ThermoFisher scientific</td>
			<td>EL0011</td>
			<td></td>
		</tr>
		<tr>
			<td>TEV</td>
			<td></td>
			<td></td>
			<td>home made</td>
		</tr>
		<tr>
			<td>TOP 10</td>
			<td>Invitrogen life technology</td>
			<td>C404003&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Euromedex</td>
			<td>26-128-3094-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Uno Q 1R column&#160;</td>
			<td>Bio-rad</td>
			<td>720 0011</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>MPBiomedicals, LLC</td>
			<td>194834</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beamline control software BsXCuBE</td>
			<td>ESRF</td>
			<td>Pernot et al. (2013), J. Synchrotron Rad. 20, 660-664</td>
			<td>local development</td>
		</tr>
		<tr>
			<td>Camserver software</td>
			<td>Dectris</td>
			<td>n.a.</td>
			<td>detector control software</td>
		</tr>
		<tr>
			<td>DAMAVER</td>
			<td>ATSAS 2.6.0</td>
			<td>http://www.embl-hamburg.de/biosaxs/download.html</td>
			<td>program to align ab initio models&#160;</td>
		</tr>
		<tr>
			<td>DAMMIF</td>
			<td>ATSAS 2.6.0</td>
			<td>http://www.embl-hamburg.de/biosaxs/download.html</td>
			<td>ab initio&#160; model reconstruction programm</td>
		</tr>
		<tr>
			<td>HPLC program Biologic Duo Flow</td>
			<td>Bio-rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC program LabSolutions</td>
			<td>Shimadzu</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>ISPyB</td>
			<td>ESRF</td>
			<td>De Maria Antolinos et al. (2015). Acta Cryst. D71, 76-85.</td>
			<td>local development</td>
		</tr>
		<tr>
			<td>PRIMUS</td>
			<td>ATSAS 2.6.0</td>
			<td>http://www.embl-hamburg.de/biosaxs/download.html</td>
			<td>program, which performs the manipulation with experimental SAXS</td>
		</tr>
		<tr>
			<td>PyMOL</td>
			<td>DeLano Scientific LLC</td>
			<td>https://www.pymol.org/</td>
			<td>software for visualization.</td>
		</tr>
		<tr>
			<td>SUPCOMB</td>
			<td>ATSAS 2.6.0</td>
			<td>http://www.embl-hamburg.de/biosaxs/download.html</td>
			<td>program to superimpose 3D structures</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioLogic Duo Flow</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioLogic Biofrac Fraction collector</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Shimadzu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labsonic P</td>
			<td>Sartorius Stedim biotech</td>
			<td>BBI8535108</td>
			<td></td>
		</tr>
	</tbody>
</table>

online size-exclusion and ion-exchange chromatography on a saxs beamline

生物小角度X射线散射（BioSAXS）是用于确定溶液的结构，颗粒大小和形状，以及表面 - 体积比大分子在分子和结构生物学的强大技术。该技术可应用于一个很宽的各种各样的溶液条件跨越大范围的浓度，pH值下，离子强度，温度，添加剂等的，但样品需要是单分散的。该警告导致了SAXS光束线液相色谱系统的实施。这里，我们描述在束线大小排阻（SEC）和离子交换色谱（IEC）的上游的整合，以获得最佳的背景减法，和数据处理不同的方法。作为一个例子，我们描述如何使用SEC-和IEC-SAXS上必不可少痘苗病毒蛋白D5的一个片段，它由一个D5N解螺旋酶域的。我们确定它的整体形状和分子量，呈现出日的六聚体结构E蛋白。

免费模式不变分析的结果列于表1中 。 D5的323-785 SEC-SAXS数据的分析显示，345 kDa的对338 kDa的分子量估计（从Porod分析），采用IEC-SAXS观察。两者都为6倍53.5 kDa的（321 kDa）的对六聚体预期质量一致。两个数据集的从头建模进行，没有强加对称性（SEC-SAXS:χ2 = 0.88，IEC-SAXS:χ2 = 3.1），使用C6对称性（SEC-SAXS:χ2 = 1.0，IEC-SAXS :χ2 = 4）。作为整体配合到两个重建散射数据是在这两种情况下（ 图1D和E）具有可比性，可以假设C6对称。因此，该模型对应于与中心通道，这似乎部分阻塞（SEC六方锥状结构: 图1F; IEC: <str翁&gt;图1G，叠加图1H）。单个模型的平均前的检查表明，该阻塞可能是的平均化处理的工件。 <img alt="图1" src="/files/ftp_upload/54861/54861fig1x.jpg" /> 图1:SAXS 数据分析和比较D5 323 -785（图改编自参考12和18）。 A）D5蛋白质和D5 323-785示意图域结构。 B）的离线离子交换色谱与三个峰的指示。橙曲线:吸光度在280nm（AU），蓝色曲线:在峰缓冲液B的缓冲液B百分比的百分比表示。 C）在线离子交换色谱。色键作为B中。此外，测得的强度在绿色指示。实验散射曲线次计算通过SEC-SAXS D）和IEC E）D5 323-785的数据分析得到的模型曲线。根据美国证券交易委员会的数据和G）IEC数据D5 323-785的F）珠模型。 高 ）美国证券交易委员会和IEC模型叠加。在F中的小图）到H）进行使用分子可视化软件24创建的。 <a href="http://ecsource.jove.com/files/ftp_upload/54861/54861fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 数据采集参数 </td><td></td></tr><tr><td>仪器: </td><td> ESRF BM29 </td></tr><tr><td>波长（埃） </td><td> 0.99 </td></tr><tr><td> Q-范围（A-1） </td><td> 0.0032 - 0.49 </td></tr><tr><td>样品到检测器的距离</td><td> 2.864米</td></tr><tr><td>曝光时间（秒） </td><td> 1每帧</td></tr><tr><td>浓度范围</td><td>呐</td></tr><tr><td>温度（K） </td><td> 293 </td></tr><tr><td>探测器</td><td>皮拉图斯1M（Dectris） </td></tr><tr><td>通量（光子/秒） </td><td> 1×10 12 </td></tr><tr><td>光束尺寸（微米2） </td><td> 700×700 </td></tr><tr><td> 为D5 323-785，SEC结构参数 </td><td></td></tr><tr><td> I 0（-1）从吉尼尔] </td><td> 0.0237 </td></tr><tr><td> RG（Å）从吉尼尔] </td><td> 48 </td></tr><tr><td> q 分钟 R G -用于吉尼尔为 Q MAX R G </td><td> 0.77 - 1.24 </td></tr><tr><td> ð 最大值 （A）[由对（R）] </td><td> 145 </td></tr><tr><td>用对于p的q范围（r）的（A -1）</td><td> 0.02 - 0.17 </td></tr><tr><td> Porod体积V P（A 3）从分散] </td><td> （570±5）×10 3 </td></tr><tr><td>分子质量为M R（kDa的）从V P] </td><td> 345 </td></tr><tr><td>从序列计算单体先生（kDa的） </td><td> 321 </td></tr><tr><td> 为D5 323-785，IEC结构参数 </td><td></td></tr><tr><td> I 0（-1）从吉尼尔] </td><td> 0.386 </td></tr><tr><td> RG（Å）从吉尼尔] </td><td> 46.5±0.1 </td></tr><tr><td> q 分钟 R G -用于吉尼尔为 Q MAX R G </td><td> 0.44 - 1.29 </td></tr><tr><td> ð 最大值 （A）[由对（R）] </td><td> 120 </td></tr><tr><td>用对于p的q范围（r）的（A -1） </td><td> 0.03 - 0.18 </td></tr><tr><td> Porod体积V P（＆＃197; 3）从分散] </td><td> （577±5）×10 3 </td></tr><tr><td>分子质量为M R（kDa的）从V P] </td><td> 339 </td></tr><tr><td>从序列计算的六聚先生（kDa的） </td><td> 321 </td></tr></tbody></table> 表1:SAXS 数据参数。表总结SAXS数据采集和分析的参数。

Watch this Scientific Journal Video about Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline at JoVE.com

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

通过小角X射线散射（SAXS）的蛋白的溶液结构的确定需要单分散样品。在这里，我们提出了两种可能性，以确保样品制备和数据采集之间的最小延迟:在线尺寸排阻色谱（SEC）和在线离子交换层析（IEC）。

在线体积排阻和离子交换色谱法SAXS光束线

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

online-size-exclusion-ion-exchange-chromatography-on-saxs

Research

JoVE Journal

Biochemistry

17.2K 次观看.  European Synchrotron Radiation Facility. 通过小角X射线散射（SAXS）的蛋白的溶液结构的确定需要单分散样品。在这里，我们提出了两种可能性，以确保样品制备和数据采集之间的最小延迟:在线尺寸排阻色谱（SEC）和在线离子交换层析（IEC）。 

视频: Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

离子交换色谱 (IEX) 与多角度光散射 (ALS) 相结合, 用于蛋白质分离和表征

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

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 ...

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Analysis of SEC-SAXS data via EFA deconvolution and Scatter

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces&#8212;chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW.

通过 EFA 去卷和散射分析 SEC-SAXS 数据

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.