这项工作是由美国国立卫生研究院授予R01 GM108635和U54 GM094618，梅奥诊所-ASU协同种子格兰特奖和NSF STC奖1231306.我们感谢A.沃克与稿件准备援助的支持。

1.过滤解决方案及血脂注意:在这个协议中使用的所有试剂和工具应该是尽可能干净，以避免引入的样品中的微粒污染物，这可能会阻塞在LCP喷射器。 <ol><li>通过过滤5微米降速过滤沉淀所有的解决方案和熔融脂质。干净注射器彻底和应用压缩空气干燥。或者，使用便携式洁净室罩，以防止在样品制备程序捕集粉尘的颗粒或纤维。 </li></ol>在LCP MP 2.重建注意:最好LCP宿主脂质结晶的选择取决于目标的MP，并且在宿主脂质筛分结晶试验14通常识别。单酰基甘油（MAG的）代表了最常见的类用于LCP结晶15脂质。所述协议是基于使用9.9 MAG（油酸单甘油酯）作为主机脂，因为这是用于消旋结晶迄今16最成功的脂质。 9.9 MAG可以通过其他的LCP宿主脂质或脂质混合物考虑到在它们的相位行为的差异之后被取代。例如，在GPCR的情况下，油酸酯通常掺杂有胆固醇受体稳定化。这里，用9:1（重量/重量）9.9 MAG:胆固醇混合物作为宿主脂质。 <ol><li>混合靶的MP，其在洗涤剂胶束溶液纯化，用使用两个注射器（＃1和＃2）和一个耦合器中的合适的LCP宿主脂质，如前面13所描述。 </li><li>简单地说，负载注射器＃1与熔融脂质和注射器＃2与MP解决方案。使用对应于这仅仅是对应的LCP宿主脂质的最大水合能力水平以下， 例如 3的脂质和水的MP溶液之间的比例:2 V 9.9 MAG / v的脂质/ MP溶液，1:1体积/体积对于大多数其他短链的MAG脂质/ MP溶液（7.9 MAG，9.7 MAG 等 ）。 </li><li>通过注射器耦合器连接的注射器，并开始通过按压柱塞向混合物来回移动针筒之间，直到样品变得均匀和透明的物质混合。制备约50微升的LCP样品用于通过LCP-SFX的完整数据集的集合。最终的样品体积通常由两（去〜100微升）约一个因素样品整合和脂质滴定（第5节）后增加。 </li></ol> 3.在注射器设置结晶<ol><li>形成透明和均一的LCP之后，移动至整个样品进入注射器＃2。取下注射器＃1，同时保持连接到注射器＃2耦合器。 </li><li>一个可移动的针（表26S）连接到另一台100微升干净的注射器（注射器＃3），吸约70微升的沉淀剂溶液进去的。沉淀剂溶液的触发f显示组合物高密度微晶的ormation为每个目标的MP唯一的，应进行到这些协议之前确定。在这里，使用收益率A 2A受体英寸（40毫米硫氰酸钠，100毫米柠檬酸钠pH值为5，26-28％PEG400）的微晶阵雨优化沉淀剂溶液组成。 </li><li>断开注射器＃3针，同时保持注射器内聚四氟乙烯密封垫圈。 </li><li>通过耦合器连接的注射器＃2和＃3，确保聚四氟乙烯套圈是正确地在两个注射器的地方。仔细拧紧耦合到位。 </li><li>东方的耦合注射器垂直注射器＃2在底部，直到LCP串触及针筒＃3柱塞从注入针筒＃2的蛋白质载货LCP样品放入注射器＃3缓慢而平稳。注入的LCP体积将达到初始沉淀体积的约1/10的注射器＃3（〜7微升）。由两个syri规模读取验证卷nges（均柱塞应约7微升移动）。 </li><li>断开注射器＃2。耦合器现在仅连接到注射器＃3包含浸在沉淀剂溶液的样品。 </li><li>用封口膜以完全密封注射器＃3，包括柱塞注射器接口，耦合器的开口端和针螺母。在该步骤中不完全密封可导致沉淀条件改变和脱水的样品。 </li><li>重复步骤3.3-3.7设立结晶6个附加注射器（＃9 4-＃）利用共有的〜LCP样品的50微升（约7微升每每个注射器的LCP）。 </li><li>储存密封注射器在一个塑料密封的袋子被一个或两个无纤维的清洁组织浸泡预用水，以防止样品脱水。晶体生长过程中密封在20℃培养箱袋和存储注射器。 </li></ol> 4.晶体检测<ol><li>从培养箱到图像在LC移除注射器P样品直接在注射器内（＃3-＃9）交叉偏振光在立体显微镜下每12-24小时。识别晶体作为紧密包装光泽粒子，或者在更小的晶体大小的情况下，作为来自LCP长丝的均匀发光。 </li><li>返回密封注射器到袋，并在20℃下储存以供将来使用。 </li></ol> 5.样品合并和滴定法7.9 MAG 注意:此处描述的脂质滴定步骤有两个目的:一个）以吸收多余的沉淀物溶液和b），以防止脂质在注射冷冻到XFEL光束。当9.9 MAG组成的LCP样品注入一个真空室，用于收集数据，集约蒸发可将样品冷却到温度低于平衡相变温度（〜18℃），将所述样品的一部分结合到一个层状结晶相（LC）。液晶相的这些修补程序，由光束击中时，产生强烈的粉DIFFR动作环，可以破坏敏感检测器6中 ，诸如康奈尔-SLAC像素阵列检测器（CSPAD）。具有较短链脂质（9.7 MAG或7.9 MAG）9.9 MAG样品的滴定缓解这个问题，因为这样的混合物具有较低的相变温度。在这里，我们描述了一个7.9 MAG美格9.9组成LCP滴定。当较短链的MAG（9.7 MAG，7.9 MAG 等 ）被用于结晶或当串行晶体数据在环境压力收集仍然需要在步骤5.11滴定，但它可以使用所采用的原LCP宿主脂质进行结晶。 <ol><li>数据集合的开始之前约1小时，取出注射器与来自20℃培养箱样品。 </li><li>选择2-4注射器基于对结晶条件类似晶体的外观和相似样品合并。 </li><li>小心取出封口膜从选定的密封注射器。 </li><li>去掉注射器耦合器和附加一个干净的可移动针（表26S）的第一选定注射器。轻缓地推动柱塞前进通过针来挤压沉淀出到一个离心管中。在此步骤练习小心，因为在此步骤中施加于柱塞的高压可以喷射一些晶体载货LCP与沉淀剂溶液一起，导致试样的部分或完全丧失。 </li><li>当大部分沉淀已被除去而LCP积累了在针入口处停止柱塞。 </li><li>重复步骤5.4-5.5与其他选定的注射器。 </li><li>为了巩固所得到的LCP样品，通过清洁耦合器两个注射器连接在一起。 </li><li>按下上一个针筒柱塞到整个样品从注射器到另一个之一转移。 </li><li>断开空的注射器。 </li><li>重复步骤5.7-5.9巩固所有晶体载货LCP材料从两个到四个预定-selected注射器到一个注射器。除去尽可能多沉淀越好。 </li><li>添加〜5微升7.9 MAG还是原来的短链MAG主机脂质到一个空的注射器。该注射器连接到通过注射器耦合器合并样品注射器和交替按压注射器活塞混合。重复，直到所有的剩余溶液被吸收并形成均匀且透明的LCP。 LCP的滴定后的最终量可以根据过量沉淀和其组合物的体积上变化，从20至35微升。 </li><li>移动整个混合样品LCP到一个注射器，并断开空的注射器。 </li></ol> 6.表征微晶<ol><li>附加一个LCP注射器针头装（点样式3，计22，长一英寸）与统一采样注射器。 </li><li>仔细喷射〜1微升在载玻片上在LCP样品，用玻璃盖玻片覆盖。轻轻按压盖玻片夹住样本。 </li><li>取的LCP样品的图像的立体显微镜下，在用亮视野照明和交叉偏振器可能的最高放大率（典型100X）。如果可能的话，使用UV-荧光显微镜和SONICC（二阶非线性成像手性晶体）17成像，以确认在样品中蛋白质微晶的存在需要额外的图像。 </li><li>估计晶体大小和密度10。用于数据收集的实验提供了理想晶体密度将取决于晶体尺寸，X射线束的直径而LCP流的直径，并应导致约10-40％的结晶的命中率。 </li></ol> 7.晶体密度调整注意:如果在步骤中找到的晶体密度6.4过高，导致多个晶体匹配的大百分比，晶体应以下来优化如下所述的步骤进行稀释样品收集数据。如果晶体密度太低对所有制备的样品在有效的数据集合，然后在晶体生长的条件，应重新优化，因为存在用于在LCP浓缩MP晶体没有可靠的方法。 <ol><li>准备所需整齐的LCP量通过模拟原始样品在LCP组合物（相同的脂质和沉淀剂）与需要进行稀释的晶体可用于稀释。 </li><li>将所有整齐LCP到一个注射器。拆下空的注射器，但留下连接耦合器。 </li><li>从包含与微晶LCP的样品取出注射器针头。样品注射器连接到通过耦合器包含整齐LCP注射器。 </li><li>通过推来回通过耦合器直到均匀性达到混合注射器的内容。 </li><li>重复第6节来重新评估调整的样品中的微晶密度。 </li></ol> 8. LCP喷油器加载第二LCP-SFX数据收集<ol><li>断开与样品注射器中的耦合器和附加一个1"加载针注射器。 </li><li>传送20-40微升样品放入一个LCP喷射器的贮存器。 </li><li>插入在LCP喷射到样品室18，启动注射器，调整流量，并收集LCP-SFX数据。 </li></ol>在LCP 9可溶性掺入蛋白质晶体注意:使用高度粘稠的介质，诸如LCP，用于递送可溶性蛋白的晶体允许一个显着减少蛋白质消耗以串行结晶实验。在这些步骤中，我们描述了如何将可溶性蛋白的晶体LCP。晶体可以通过任何技术来获得，在晶体悬浮液的形式合并在一起，并在必要时进行过滤。 <ol><li>调整晶体密度悬浮以保证约10-40％结晶命中率。 </li><li> ŧEST使用LCP使用7.9 MAG，9.7 MAG或1的沉淀剂溶液的相容性:7.9 MAG的1:1混合物和9.9 MAG作为宿主脂质。 </li><li>混合〜25微升晶体悬浮液的〜25微升用注射器混频器，直至均匀LCP形式主机脂质。 </li><li>如第6装入样品在LCP注射器描述并收集数据，如第8描述评估晶体大小和密度。 </li></ol>

<ol>
	<li>Cherezov, V., 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=High-resolution+crystal+structure+of+an+engineered+human+beta2-adrenergic+G+protein-coupled+receptor.">High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.</a> Science. 318 (5854), 1258-1265 (2007).</li><li>Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidic+cubic+phase+technologies+for+membrane+protein+structural+studies.">Lipidic cubic phase technologies for membrane protein structural studies.</a> Curr Opin Struc Biol. 21 (4), 559-566 (2011).</li><li>Chapman, H. N., 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=Femtosecond+X-ray+protein+nanocrystallography.">Femtosecond X-ray protein nanocrystallography.</a> Nature. 470 (7332), 73-77 (2011).</li><li>Neutze, R., Wouts, R., van der Spoel, D., Weckert, E., Hajdu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+for+biomolecular+imaging+with+femtosecond+X-ray+pulses.">Potential for biomolecular imaging with femtosecond X-ray pulses.</a> Nature. 406 (6797), 752-757 (2000).</li><li>DePonte, D. 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=Gas+dynamic+virtual+nozzle+for+generation+of+microscopic+droplet+streams.">Gas dynamic virtual nozzle for generation of microscopic droplet streams.</a> J Phys D Appl Phys. 41 (19), 195505 (2008).</li><li>Weierstall, U., 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=Lipidic+cubic+phase+injector+facilitates+membrane+protein+serial+femtosecond+crystallography.">Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography.</a> Nat Commun. 5, 3309 (2014).</li><li>Fenalti, G., 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=Structural+basis+for+bifunctional+peptide+recognition+at+human+&#948;-opioid+receptor.">Structural basis for bifunctional peptide recognition at human &#948;-opioid receptor.</a> Nat Strcut Mol Biol. 22 (3), 265-268 (2015).</li><li>Liu, W., 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=Serial+femtosecond+crystallography+of+G+protein-coupled+receptors.">Serial femtosecond crystallography of G protein-coupled receptors.</a> Science. 342 (6165), 1521-1524 (2013).</li><li>Zhang, 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=Structure+of+the+Angiotensin+Receptor+Revealed+by+Serial+Femtosecond+Crystallography.">Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography.</a> Cell. 161 (4), 833-844 (2015).</li><li>Liu, W., Ishchenko, A., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+microcrystals+in+lipidic+cubic+phase+for+serial+femtosecond+crystallography.">Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography.</a> Nat Protoc. 9 (9), 2123-2134 (2014).</li><li>Liu, W., 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=Structural+basis+for+allosteric+regulation+of+GPCRs+by+sodium+ions.">Structural basis for allosteric regulation of GPCRs by sodium ions.</a> Science. 337 (6091), 232-236 (2012).</li><li>Caffrey, M., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallizing+membrane+proteins+using+lipidic+mesophases.">Crystallizing membrane proteins using lipidic mesophases.</a> Nat Protoc. 4 (5), 706-731 (2009).</li><li>Liu, W., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+membrane+proteins+in+lipidic+mesophases.">Crystallization of membrane proteins in lipidic mesophases.</a> J Vis Exp. (49), (2011).</li><li>Li, D., Shah, S. T. A., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+Lipid+and+Temperature+as+Important+Screening+Variables+for+Crystallizing+Integral+Membrane+Proteins+in+Lipidic+Mesophases.+Trials+with+Diacylglycerol+Kinase.">Host Lipid and Temperature as Important Screening Variables for Crystallizing Integral Membrane Proteins in Lipidic Mesophases. Trials with Diacylglycerol Kinase.</a> Cryst Growth Des. 13 (7), 2846-2857 (2013).</li><li>Caffrey, M., Lyons, J., Smyth, T., Hart, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoacylglycerols:+The+Workhorse+Lipids+for+Crystallizing+Membrane+Proteins+in+Mesophases.">Monoacylglycerols: The Workhorse Lipids for Crystallizing Membrane Proteins in Mesophases.</a> Current Topics in Membranes. 63, (2009).</li><li>Kulkarni, C. V., Wachter, W., Iglesias-Salto, G., Engelskirchen, S., Ahualli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoolein:+a+magic+lipid?.">Monoolein: a magic lipid?.</a> Phys Chem Chem Phys. 13 (8), 3004-3021 (2011).</li><li>Kissick, D. J., Gualtieri, E. J., Simpson, G. J., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+optical+imaging+of+integral+membrane+protein+crystals+in+lipidic+mesophases.">Nonlinear optical imaging of integral membrane protein crystals in lipidic mesophases.</a> Anal Chem. 82 (2), 491-497 (2010).</li><li>James, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Injection Methods and Instrumentation for Serial X-ray Free Electron Laser Experiments. , 1-126 (2015).</li><li>Kang, Y., 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+rhodopsin+bound+to+arrestin+by+femtosecond+X-ray+laser.">Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.</a> Nature. 523 (7562), 561-567 (2015).</li><li>Rasmussen, S. G. 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=Crystal+structure+of+the+&#946;2+adrenergic+receptor-Gs+protein+complex.">Crystal structure of the &#946;2 adrenergic receptor-Gs protein complex.</a> Nature. 477 (7366), 549-555 (2011).</li><li>Fromme, R., 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=Serial+femtosecond+crystallography+of+soluble+proteins+in+lipidic+cubic+phase.">Serial femtosecond crystallography of soluble proteins in lipidic cubic phase.</a> IUCrJ. 2 (Pt 5), 545-551 (2015).</li><li>Sugahara, 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=Grease+matrix+as+a+versatile+carrier+of+proteins+for+serial+crystallography.">Grease matrix as a versatile carrier of proteins for serial crystallography.</a> Nat Meth. 12 (1), 61-63 (2014).</li><li>Conrad, C. 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=A+novel+inert+crystal+delivery+medium+for+serial+femtosecond+crystallography.">A novel inert crystal delivery medium for serial femtosecond crystallography.</a> IUCrJ. 2 (Pt 4), 421-430 (2015).</li><li>Botha, 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=Room-temperature+serial+crystallography+at+synchrotron+X-ray+sources+using+slowly+flowing+free-standing+high-viscosity+microstreams.">Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams.</a> Acta Crystallogr Sct D. 71 (Pt 2), 387-397 (2015).</li><li>Nogly, 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=Lipidic+cubic+phase+serial+millisecond+crystallography+using+synchrotron+radiation.">Lipidic cubic phase serial millisecond crystallography using synchrotron radiation.</a> IUCrJ. 2, 168-176 (2015).</li><li>Tenboer, J., 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+serial+crystallography+captures+high-resolution+intermediates+of+photoactive+yellow+protein.">Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein.</a> Science. 346 (6214), 1242-1246 (2014).</li><li>Kupitz, C., Basu, S., Grotjohann, I., Fromme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+time-resolved+crystallography+of+photosystem+II+using+a+femtosecond+X-ray+laser.">Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser.</a> Nature. 513, 261-265 (2014).</li></ol>

The authors have no conflicts of interest to disclose.

这里所描述的协议提供了一个标准的LCP-SFX实验用的LCP注射器的样品制备过程的概要。根据我们的经验，用于收集完整数据集所需的最终微晶载货的LCP样品的总量典型地是50-100微升（ 表1; 25-50微升初始蛋白质载货的LCP样品的），这取决于微晶大小，质量和密度。由于每个100μl的气密玻璃注射器可容纳约7微升的LCP处于完全扩展字符串的形式，以确保适当甚至沉淀剂溶液进LCP的扩散，在注射器至少四个到七个样品应来制备每个LCP-SFX实验。 由于样品通过窄20-50微米的直径的毛细管中挤出，这是至关重要的，以避免在样品中的任何外来微粒材料。可能在LCP不定期地推出了粉尘和纤维样品或沉淀剂溶液可能堵塞注射器毛细管和在实验过程中增加了停机时间。因此，制备样品之前过滤通过5μm微孔过滤脂质和所有的解决方案是非常重要的。任选地，清洁室或便携式洁净室罩可用于样品制备。 这些协议是基于使用9.9 MAG（油酸单甘油酯）作为宿主脂质为MP的结晶。然而，它们不限于9.9 MAG，并且可以考虑到在脂质相行为的差异之后很容易地适应于任何其他的LCP主机脂质或脂质混合物。大多数在LCP中结晶国会议员的结构制得使用的MAG之一，以9.9的MAG是最成功的代表迄今15。使用9.9 MAG为SFX的主要缺点是在冷却低于18℃时，当在真空中进行数据采集时经常发生其向层状结晶相变。该titrati与7.9 MAG在本协议中的第5节提供了一个很好的解决这个问题。根据我们的经验，水晶承受无任何不良影响这样的剂量调整。然而，如果添加7.9 MAG或另一个短链MAG的是不可取的，滴定可以用9.9的MAG来执行。在这种情况下，稳定的LCP注射期间流入真空气体应从氦被切换成氮气，它可以防止结晶脂质相的大多数样品中的形成，在具有较不稳定的试样流的费用。 LCP的凝胶状稠度具有很大的优势，其用作晶载体介质，因为其允许在宽范围的晶体流量，适用于串行晶体的数据收集在现代XFEL和同步加速器源的调整。数量级的C订单匹配与XFEL脉冲重复率导致急剧减少晶体消费晶体流量ompared液体注入。 LCP是用于递送不仅在它生长的MP晶体合适的载体介质，也为可溶性蛋白21的晶体。几种可供选择的粘稠晶系媒体最近引入了22 - 24，延长工具和试剂的阿森纳进行连续结晶实验。此外，使用LCP作为晶体递送介质串行晶体已被证明在常规同步加速器源中，至少井衍射晶体24,25。该升压的X射线数量级的强度，以及改善在检测器技术的同步加速器源未来的升级，将使串行晶体更加方便和有吸引力的结构测定方法。 LCP-SFX已经证明其对MP结构测定效力。对于具有挑战性的生物系统（如GPCR的或大分子复合物），其中lARGE，衍射品质晶体是难以生长，LCP-SFX可提供一个有吸引力的，如果不是唯一的，可行的选择来解决的原子分辨率结构。其所有的优点， 即使用LCP作为基质为MP的结晶和晶体递送，低蛋白消耗，不存在辐射损伤，室温收集数据，时间分辨研究26,27的可能性，没有晶体收获要求，LCP- SFX应在结构生物学的未来产生重要的作用。

X射线晶体是最成功的技术，迄今为解决膜蛋白（MPS）的原子分辨率的结构。从脂质中间相，也被称为脂质立方相（LCP），或在消旋结晶，结晶表示在MP晶体已启用的具有挑战性的目标的高分辨率结构确定诸如G蛋白偶联受体的主要发展酮（GPCR的）1。在LCP工具和技术的最新发展，使这一技术提供给大型社区在世界各地的2结构生物学家的。然而，在许多情况下，在LCP形成MP的结晶太小，可以使用即使在最先进的微聚焦同步加速器束线，其中所述样品从严重辐射损伤和劣化遭受之前可以得到3在高分辨率足够的信号。 一种新的方法，以晶体数据收集是与第一X射线自由电子激光（XFEL）的调试启用。该方法是基于"毁灭前的衍射"的原则，首先介绍了理论上4，然后通过实验证实了在直线加速器相干光源（LCLS）3，全球硬XFELs先驱生物样品。一个XFEL几十飞秒的持续时间，从一个原始晶体前蛋白原子可以响应​​于辐射损伤移动衍射内产生高亮度的X射线脉冲。在最初的实验中，微晶连续在通过液体喷射器5产生的含水的流供给到与XFEL波束的交叉点。该实验装置被称为飞秒系列晶体（SFX）。使用液体喷射器的SFX的主要缺点是它们的流动速率高，这因而需要几十到几百的完整数据集的收集纯化蛋白的毫克。通过EMPloying一个特殊注射器，可以处理凝胶状的LCP样品（LCP microextrusion喷油器）6，我们成功地传递在一个LCP基质生长的几种不同的人的GPCR的微晶并获得它们的高分辨率结构6 - 9，具有显著减少蛋白质消费每数据集下0.3毫克。喷射器由可以容纳晶体载货LCP高达20,40或100微升并通过该样品通过高压应用挤出（高达10,000窄毛细管（10-50微米直径）的贮存器的PSI）从液压柱塞与压力扩增阶段，通过从HPLC（高效液相色谱法）泵的液体驱动。 LCP的离开毛细管喷嘴的流由非反应性气体（一般为氦或氮）的平行流稳定化。 在这里，我们提供了所需的步骤视觉演示准备和表征样本对于LCP-SFX实验。其他详细信息可以在公布的协议10被发现。作为一个例子，我们将使用腺苷A 2A受体11，准备这种蛋白质的晶体LCP使用SFX方法X射线数据收集。而我们的协议是与能够由在XFEL和同步加速器源串行晶体流用于数据收集的LCP的任何注射器兼容，为了说明的目的，我们将使用在文献中描述的LCP喷射器。 6.产生LCP高密度微晶优化沉淀条件应通过高通量的结晶在进行此协议之前筛选12,13确定。此协议的典型流程图如图1所示。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>LCP host lipid monoolein (9.9 MAG)</td>
			<td>Nu Chek Prep</td>
			<td>M239</td>
			<td>Hosting lipid for the protein to be crystallized</td>
		</tr>
		<tr>
			<td>LCP host lipid monoolein (9.9 MAG)</td>
			<td>Sigma</td>
			<td>M7765&#160;</td>
			<td>Hosting lipid for the protein to be crystallized</td>
		</tr>
		<tr>
			<td>LCP host lipid monopalmitolein (9.7 MAG)</td>
			<td>Nu Chek Prep</td>
			<td>M219</td>
			<td>Hosting lipid for the protein to be crystallized</td>
		</tr>
		<tr>
			<td>LCP host lipid 7.9 MAG</td>
			<td>Avanti Polar Lipids</td>
			<td>850534</td>
			<td>Hosting lipid for tiration to avoid lamellar crystal phase (Lc) formation during sample injection</td>
		</tr>
		<tr>
			<td>Purified protein solution concentrated to 20-50 mg/mL</td>
			<td></td>
			<td></td>
			<td>Target protein</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C606-1</td>
			<td>Used to dissolve lipids</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>179337-500ML</td>
			<td>Used to wash syringes</td>
		</tr>
		<tr>
			<td>Eleven 100 mL Hamilton gas-tight syringes without needles</td>
			<td>Hamilton</td>
			<td>7656-01</td>
			<td>For LCP preparation</td>
		</tr>
		<tr>
			<td>Eight syringe couplers</td>
			<td>Hamilton</td>
			<td>SKU 209526</td>
			<td>For LCP preparation</td>
		</tr>
		<tr>
			<td>Removable flat-tipped needle</td>
			<td>Hamilton</td>
			<td>7804-01</td>
			<td>For LCP dispensing</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Parafilm</td>
			<td>M&#39;, 250&#39; x 2&#34;</td>
			<td>Used for syringe sealing</td>
		</tr>
		<tr>
			<td>Lint free lens cleaning tissues</td>
			<td>Fisher</td>
			<td>NC9592151</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal filter tubes, Ultrafree-MC, 0.5 mL, Durapore 5 &#181;m</td>
			<td>Millipore</td>
			<td>UFC3 0SV 00</td>
			<td>Used for filtering of precipitant solutions and lipids</td>
		</tr>
		<tr>
			<td>Microscope slides, 1 inch x 3 inch&#160;</td>
			<td>GoldSeal</td>
			<td>3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Two wash bottles with fine tips. One filled with milli-Q or distilled water, the other with methanol</td>
			<td>VWR</td>
			<td>89094-640</td>
			<td>These are used to clean the syringes, needles and coupler.&#160; Methanol is used first, then water.</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>Microflex</td>
			<td>XC-310-L</td>
			<td>For blow drying syringes, needles, ferrules, and couplers</td>
		</tr>
		<tr>
			<td>Safety glasses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pressurized air cans</td>
			<td>Office Depot (Dust-Off)</td>
			<td>527494</td>
			<td>For syringe cleaning</td>
		</tr>
		<tr>
			<td>Dry block heater with a digital temperature controller</td>
			<td>Fisher</td>
			<td>11-720-10BQ&#160;</td>
			<td>Used for thawing lipids</td>
		</tr>
		<tr>
			<td>Benchtop Centrifuge&#160;</td>
			<td>Fisher</td>
			<td>05-413-340</td>
			<td>Spinning down solutions</td>
		</tr>
		<tr>
			<td>Clean Room Mini - 12 inch Portable Positive Pressure Hood with HEPA filter</td>
			<td>Sentry Air Systems, Inc.</td>
			<td>SS-112-PCR</td>
			<td>Cleaning sample preparation</td>
		</tr>
		<tr>
			<td>Stereo zoom microscope equipped with a linear polarizer and rotating analyzer&#160;</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
			<td>Crystal density check</td>
		</tr>
		<tr>
			<td>UV microscope</td>
			<td>JAN Scientific</td>
			<td>UVEX-P</td>
			<td>Optional, for crystal imaging purposes</td>
		</tr>
		<tr>
			<td>SHG imager&#160;</td>
			<td>Formulatrix</td>
			<td>SONICC</td>
			<td>Optional, for crystal imaging purposes</td>
		</tr>
	</tbody>
</table>

preparation and delivery of protein microcrystals in lipidic cubic phase for serial femtosecond crystallography

膜蛋白（MPS）是细胞膜和初级药物靶的基本成分。合理的药物设计依赖于精确的结构的信息，典型地通过结晶得到的;然而，国会议员都难以结晶。在MP结构测定的最新进展已经从脂质立方相（LCP）的结晶方法，通常产生良好的衍射，但往往小的晶体是从同步加速器源的传统晶体数据收集过程中辐射损伤遭受的发展中受益匪浅。新一代的X射线自由电子激光（XFEL）产生极其明亮的飞秒脉冲源的发展，使室内温度数据采集从没有或可忽略不计的辐射损伤微晶。我们最近在LCP技术与串行飞秒晶体（LCP-SFX）相结合的努力已导致数人G蛋白偶联受体的高分辨率结构，WHICH代表结构测定一个非常困难的目标。在LCP-SFX技术，LCP被招募为经济增长和交付MP微晶到喷射流的交汇与XFEL梁晶体数据收集的矩阵。它已被证实LCP-SFX可以大幅度提高衍射分辨率时仅子10微米的晶体是可用的，或当在室温下使用较小的晶体可以克服与较大制冷机冷却的结晶相关的各种问题，如缺陷积聚，高mosaicity和cryocooling文物。在X射线源和检测器技术的未来进步应使串行晶体不仅在XFELs，而且在更方便同步加速器束线高度吸引力和可行的实施。在这里，我们提出详细的视觉协议串行结晶实验制备，表征和微晶交付LCP。这些协议包括用于注射器进行结晶实验，检测和表征的晶体样品，优化晶体密度，装载微晶载货LCP到注射器装置和样品递送至光束收集数据的方法。

下面我们描述了按照上述方案制备的样品，使我们能够收集SFX数据和解决五人GPCR的高分辨率结构得到代表性的结果:在复杂的血清素受体​​5-HT 2B与激动剂麦角胺8（PDB ID 4NC3 ），平滑受体（SMO）在复杂的拮抗剂环巴胺6（PDB ID 4O9R），在复合δ阿片受体在复杂的双功能肽配位体磷酰化-NH 2 7（PDB ID 4RWD），血管紧张素受体 ​​具有阻滞剂ZD7155 9（PDB ID 4YAY），和视紫红质和arrestin的19（PDB ID 4ZWJ）之间的复合物;和两个测试可溶性蛋白:溶菌酶（PDB ID 4ZIX）和藻蓝蛋白（PDB ID 4ZIZ）。 5-HT 2B介导的神经递质血清素的各中央和周边的生理功能。这种受体被用于建立和验证在LCP-SFX方法，通过比较在LCLS与先进光子源（APS）由传统microcrystallography解决的制冷机冷却的结构中得到的室温下的结构。总共用蛋白微晶100μl的LCP样品（平均粒径约5μm）的〜制备并用于SFX数据收集，以及5HT 2B的LCP-SFX结构在2.8埃（ 图2）8被成功地解决。在表1中被设置在样品制备和数据收集的其他有用的信息。 平滑化受体（SMO）是F级的GPCR的成员和致畸环巴胺的分子靶标，在胚胎发育和肿瘤生长良好的证明函数。最初，获得具有120×10×5微米的平均尺寸SMO /环巴胺的相对大的晶体，然后用于数据collecti就在同步辐射光源使用传统的基于测角仪，晶体。然而，这些大的晶体产生的在从大mosaicity患（上述2-3度），这可能是由于晶体生长缺陷的积累，或从与cryocooling效果的微焦点光束线（10微米直径）衍射差。 LCP-SFX，但是，使我们在LCLS从在室温下5微米大小的晶体，收集质量衍射数据。其结构通过分子置换在各向异性3.4，3.2和4.0埃分辨率沿着三个主轴解决，明确地识别所述结合口袋6（ 图3）的环巴胺的位置。 生物碱鸦片制剂，例如吗啡，靶向μ阿片样物质受体（μ-OR）被广泛地用于治疗严重疼痛管理。然而，他们的广泛使用导致收购耐受性和成瘾性。吗啡类药物的联合给药用δ阿片受体（δ-OR）拮抗剂已经显示出，以防止上述的副作用，从而促进了混合δ型或拮抗剂和μ或激动剂功能的化合物的搜索。我们得到在δ型或与双官能四肽磷酰化的NH 2的复合使用衍射到3.3埃在使用常规基于测角数据收集策略同步加速器X射线源制冷机冷却的晶体的初始衍射数据。这些数据揭示了肽配体的局部模糊的电子密度。随后，获得在室温下XFEL衍射数据，并以2.7埃的分辨率表示为配体和受体7清晰密度测定的结构。这种结构提供了进一步了解阿片受体功能和选择性的机会和新的镇痛药的发展提供了宝贵的见解。 1 R）是一个GPCR作为血压的主要调节剂。我们结晶AT 1 R在复杂与LCP拮抗剂ZD7155。优化晶体达到了40×4×4微米3的最大尺寸最好的衍射在同步辐射光源只达到〜4。通过改变结晶条件，我们获得了更小的晶体阵雨（10×2×2微米3），它被用来在使用XFEL辐射2.9的决议获得室温结构。共有2764739探测器图像采集，使从对应于约0.29毫克蛋白9的65微升水晶载货LCP的完整数据集。帧的总数目，457275被鉴定为晶体匹配，对应于17％的命中率，其中73130帧（命中的16％）进行了成功地索引和集成。 GPCR的信号通过任何的G蛋白或抑制蛋白介导的两个主要途径。结合于异克S-蛋白的β2 -肾上腺素能受体的结构，几年前20解决了，而一个GPCR的与休止复杂结构已仍然难以捉摸。我们获得了一个LCP视紫红质抑制蛋白融合蛋白达到25-30微米最长尺寸小晶体，但尽管进行了广泛的优化，只衍射在同步加速器光源〜7的分辨率。通过使用LCP-SFX方法，12小时XFEL beamtime内，我们收集了22262水晶命中，其中有18874模式被成功收录并整合到各向异性3.8 / 3.8 / 3.3 19的分辨率极限。视紫红质抑制蛋白是一个非常具有挑战性的蛋白复合物，使用传统的方法抵制结构的决心。这种结构的成功判定已经证明的巨大潜力在LCP-SFX方法解决难题，并提供检查休止偏置信号在G蛋白偶联受体机制的独特机会。 最后，除了在膜中的脂类相生长的蛋白质晶体，我们的样品制备和递送方法成功适于可溶性蛋白质晶体，其中LCP用作晶体的递送的载体介质，使我们能够显着地降低了蛋白质消费所需的结构测定。两型蛋白，溶菌酶和藻蓝蛋白的结构，分别在1.89埃与1.75埃分辨率分别用这种方法解决，使用低于0.1毫克每蛋白21的。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> 五羟色胺受体2B </td><td> 磨平受体 </td><td><strong&gt;δ阿片受体</td><td> 血管紧张素II受体1型 </td><td> 视紫红质抑制蛋白 </td><td> 溶菌酶 </td><td> 藻蓝蛋白 </td></tr><tr><td> PDB ID </td><td> 4NC3 </td><td> 4O9R </td><td> 4RWD </td><td> 4YAY </td><td> 4ZWJ </td><td> 4ZIX </td><td> 4ZIZ </td></tr><tr><td>共抽查使用*，微升</td><td> 100 </td><td> 83 </td><td> 50 </td><td> 65 </td><td> 75 </td><td> 10 </td><td> 10 </td></tr><tr><td>用总蛋白，微克</td><td> 300 </td><td> 500 </td><td> 300 </td><td> 290 </td><td> 340 </td><td> 100 </td><td> 100 </td></tr><tr><td>平均晶体尺寸，微米</td><td> 5×5×5 </td><td> &lt;5 </td><td> 5×2×2 </td><td> 10×2×2 </td><td> 5-10 </td><td> 5×2×2 </td><td> 10×10×5 </td></tr><tr><td>命中率， ％ </td><td>3.6 </td><td> 7.8 </td><td> 5.9 </td><td> 17 </td><td> 0.45 </td><td> 40 </td><td> 6.5 </td></tr><tr><td>总的数据采集时间，小时</td><td> 10 </td><td> 8 </td><td> 4.6 </td><td> 6.4 </td><td> 12 </td><td> 0.75 </td><td> 0.67 </td></tr><tr><td>索引模式数</td><td> 32819 </td><td> 61964 </td><td> 36083 </td><td> 73130 </td><td> 18874 </td><td> 54,544 </td><td> 6629 </td></tr><tr><td>空间群</td><td> C 2 2 2 1 </td><td> P 2 1 </td><td> C 2 </td><td> C 2 </td><td> P 2 1 2 1 2 1 </td><td> P 4 3 2 1 2 </td><td> H 3 2 </td></tr><tr><td>分辨率， </td><td> 2.8 </td><td> 3.2 / 3.4 / 4.0 </td><td> 2.7 </td><td> 2.9 </td><td> 3.3 / 3.8 / 3.8 </td><td> 1.89 </td><td> 1.75 </td></tr></tbody></table> 表1为代表的结构的样品制备和数据收集统计汇总使用LCP-SFX方法来解决。 样品为一个LCP-SFX实验所需的量取决于晶体衍射质量，结晶密度，喷射器喷嘴的直径，以及所述XFEL光束尺寸，强度和脉冲重复率。 <img alt="图1" src="/files/ftp_upload/54463/54463fig1.jpg" /> 图1 为LCP-SFX。样品制备的典型的样品制备过程的流程图开始于在气密玻璃注射器靶蛋白的LCP结晶。得到的结晶后，将结晶载货LCP固结在一个注射器中，并用另外的脂质滴定，至absorb将多余的沉淀剂溶液。水晶XFEL使用数据采集之前，各种显微镜方法，然后表征。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54463/54463fig1large.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/54463/54463fig2.jpg" /> 图 2. 代表性的结果，结构5-HT 2B 与麦角胺复杂。（ 一 ）5-HT 2B /麦角胺微晶使用亮场显微镜模式8成像。该图已被从参考8与来自科学版权许可重复使用。（B）微晶5-HT 2B /麦角胺的使用交叉偏光显微镜模式8成像。这个数字已经从参考8版权重用许可科学（C）由LCP-SFX方法获得的5-HT 2B /麦角胺结构的卡通代表。建在模型中的脂质显示在棒表示。该配体麦角胺示于球体表示。实线表示近似膜的界限。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54463/54463fig2large.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/54463/54463fig3.jpg" /> 图3 的代表性结果，SMO在复杂的结构与环巴胺，左面板，SMO /环巴胺的微晶使用交叉偏光显微镜模式6成像。这个数字已经从与大自然沟通版权许可参考6重用。右侧面板上，一个卡通representati由LCP-SFX方法获得的SMO /环杷结构。配体环杷示于球体表示。实线表示近似膜的界限。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54463/54463fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography at JoVE.com

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

制备及脂质立方相反应蛋白微晶交付串行飞秒晶体

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

preparation-delivery-protein-microcrystals-lipidic-cubic-phase-for

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.4K 次观看.  University of Southern California. We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

视频: Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

自动声胶肽受体激动剂的效价测定测定的连续稀释

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

科研

生物化学

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

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 ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

用于大分子 X 射线晶体学的一体式样品支架,具有最小的背景散射

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.