我们想感谢曼弗雷德Kriechbaum Hecus XRS和在奥地利格拉茨的奥地利科学院生物物理研究所和纳米系统研究。 LL和XW部分由美国能源部支持下，NERI-C号DE-FG07-07ID14889奖，美国核管理委员会，根据奖励编号:NRC-38-08-950。部分由美国能源部也支持SWAXS仪器，在裁决号DE-NE0000325。

1。的样品的制备<ol><li>使用一根针，以除去一些样品从样品容器。 </li><li>使用的针，以填补在毛细管（最大直径为2.2毫米）用样品。的毛细管必须填写从底部2和3厘米之间。 </li><li>关闭的毛细血管，熔蜡器在前端。 </li><li>拧下从系统的真空样品架。 </li><li>以毛细管融合的结束（蜡年底），将未熔的样品架。 </li><li>持有人将到系统中，螺杆的地方。 </li></ol> *固体样品（包括粉末样品），可以直接放置在样品架（没有毛细必要），而液体样品必须被放置在一个毛细管。 启动SWAXS机 2。源冷启动程序<ol><li>在推动安全快门按钮，关闭快门。 </li><li>切换主电源按绿色按钮上的"ON"。 </li><li>验证紧急"关闭"按钮，在延伸的位置。 </li><li>按下绿色的按钮切换主电源。 </li><li>确认的联锁LED灯是绿色，表示所有的互锁确定的。 </li><li>等待在触摸屏上加载。它加载后，按"R6"菜单上的。 </li><li>关闭待机键，"ON"的位置。 </li><li>转动透视关键的"ON"位置。红色的X-射线灯应点亮。 </li><li>等待读取待机的水平（30千伏及0.4毫安）的触摸屏。 </li><li>将备安全的关键在"OFF"的位置。按下"启动循环"的按钮，在触摸屏的底部。 </li><li>等待在触摸屏上读额定功率（50千伏和1 mA）。如果使用不同的功率电平，进入配置屏幕，在触摸屏上按"R4"，并输入所需的设置。 </li></ol> 3。冷水机程序<ol><li>打开电源SWITCH在温度控制面板上的"ON"位置。的控制灯和LED温度显示器会亮起。 </li><li>打开电源开关至"ON"的位置上冷水机组。 </li><li>等待，直到温度显示屏显示"OFF"。这是待机模式。 </li><li>按下并按住ENTER按钮（返回符号）约4秒或直到显示的温度指示的实际温度。 </li><li>要改变温度，按向上或向下的箭头。将会显示在温度显示为约8秒的设定值之前，它会返回到的实际值。 </li><li>一旦所需的设定值显示，按ENTER键。这将存储该值。 </li><li>等待，直到实际温度达到所需的设定值。 </li></ol> 注意:如果在任何时候出现错误"E 01"显示的温度显示，按照冷水机组停机指令，填充浇注罐过滤的纯净水洗澡单位l，然后执行冷水机组一次启动程序。 4。关闭冷水机组<ol><li>按"输入"键约4秒。 </li><li>打开电源开关到"OFF"的位置。 </li></ol> 5。打开真空<ol><li>确保真空门被固定就位。 </li><li>真空1和2旋钮转到"ON"位置。 </li><li>等待，直到VAP5真空表的读数小于1.5毫巴的真空度。 </li></ol> 6。检测器的系统设置<ol><li>要设置的气体压力，验证主压力表的减压阀是在至少10巴。 </li><li>打开主阀。 </li><li>慢慢地打开，直到压力达到工作压力阀的工作压力表的8栏。 </li><li>打开第二个主阀</li><li>调整气体的流动，打开主配压阀，气体控制面板上通过转动旋钮到垂直位置。 </li><li>调整流量针型阀，直到压力表读数率约8巴。 </li><li>打开主电源开关至"ON"的位置。 LED灯应点亮。 </li><li>要调整高电压，高电压激活的开关旋转到"ON"的位置。 LED灯应点亮。 </li><li>慢慢转动约3.5KV电压控制旋钮，直至达到压力表上。不要超过4KV。 </li></ol> 7。校准<ol><li>以上的实验步骤进行与样品的已知峰。 </li><li> ASA 3.3用于获得图5峰的强度与通道。 </li><li>进入功能表→"选项"→输入通道和相应的强度。 </li><li> ，转到ASA3，"TPF"选项卡。 </li><li>更改过滤器，左上方1毫米镍（束过滤器）。 </li><li>设置位置32,000（28,000至32,000范围内，大约28000名的临界区）→位置，确保真空度低于5毫巴，确保检测是原因丁1K-10K之间。 </li><li>运行，持续10秒，每秒约52计数。 </li><li>更改能源窗，（或只与样品，不包括过滤器的情况下测量的能量窗）。将最后的奔跑，持续10秒。 </li><li>查找的位置的主光束（例如信道233）。 </li><li>对于其他的运行，更改过滤器再次到2毫米钨（W，梁挡块），从30000开始。 </li><li>查找其他所有的样品峰的位置。 </li><li>使用ImageJ的宏的软件校准SAXS -从强度与信道转换的强度与q（下或d间距）（ 图1）。 </li><li>使用ImageJ的宏软件校准WAXS - ，从强度与信道转换的强度与q（下或d间距）（ 图2）。 </li></ol> 8。软件程序<ol><li>使用该软件ASA 3.3，收集实时数据，实时数据"→"和TPF选项卡→"保存文件。 </li><li>对于EasySWAXS，点击"设备设置"选项卡。 </li><li>输入的"a"，λ，和d的值，以及重心。 </li><li>选择点瞄准。 </li><li>选择球状粒子类型（除非它是公知的是扁平状颗粒的杆）。 </li><li>按一下的吉尼尔情节"选项卡上。的I和Q 2的图形将被显示出来。 </li><li>拖动垂直线包围的部分近似直线的斜率。 </li><li>在屏幕的底部，R值会被显示。这是一个验证的要求。 </li><li>继续拖动垂直线更接近在一起，直到验证值是1和2之间。这是主观的。的R值，或回转半径，是一个估计。 </li></ol> 9。来源关机程序<ol><li>验证的安全性和快速的快门关闭。 </li><li>关闭待机键，"ON"的位置。 </li><li>等待所述触摸屏来读取备用水平。 </li><li>按R5的touchscreen。 </li><li>降低电流到0 A. </li><li>的电压降低到0 A。 </li><li>转动透视键的"OFF"位置。 </li><li>通过按下红色的关闭按钮，切换主电源"OFF"。 </li></ol>

<ol>
	<li>Bernad&#243;, P., Blackledge, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology:+Proteins+in+dynamic+equilibrium.">Structural biology: Proteins in dynamic equilibrium.</a> Nature. 468, 1046-1048 (2010).</li><li>Zhang, F., Skoda, M. W. A., Jacobs, R. M. J., Martin, R. A., Martin, C. M., Schreiber, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Interactions+Studied+by+SAXS:+Effect+of+Ionic+Strength+and+Protein+Concentration+for+BSA+in+Aqueous+Solutions.">Protein Interactions Studied by SAXS: Effect of Ionic Strength and Protein Concentration for BSA in Aqueous Solutions.</a> J. Phys. Chem. B. 111, 251-259 (2007).</li><li>Maranas, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+environment+on+local+dynamics+of+macromolecules.">The effect of environment on local dynamics of macromolecules.</a> Current Opinion in Colloid &amp; Interface Science. 12, 29-42 (2007).</li><li>Stribeck, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> X-Ray Scattering of Soft Matter. , (2007).</li><li>Mertens, H. D., 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=Structural+characterization+of+proteins+and+complexes+using+small-angle+X-ray+solution+scattering.">Structural characterization of proteins and complexes using small-angle X-ray solution scattering.</a> J. Struct. Biol. 172 (1), 128 (2010).</li><li>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=Small-angle+X-ray+and+neutron+scattering+as+a+tool+for+structural+systems+biology.">Small-angle X-ray and neutron scattering as a tool for structural systems biology.</a> Biol. Chem. 391 (7), 737 (2010).</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> Quat. Rev. Biophys. 40, 191-285 (2007).</li><li>Bonini, M., Fratini, E., Baglioni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAXS+study+of+chain-like+structures+formed+by+magnetic+nanoparticles.">SAXS study of chain-like structures formed by magnetic nanoparticles.</a> Materials Science &amp; Engineering C-Biomimetic and Supramolecular Systems. 27 (5-8), 1377-1381 (2007).</li><li>Falletta, E., Ridi, F., Fratini, E., Vannucci, C., Canton, P., Bianchi, S., Castelvetro, V., Baglioni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tri-block+copolymer+templated+synthesis+of+gold+nanostructures.">A tri-block copolymer templated synthesis of gold nanostructures.</a> Journal of Colloid and Interface Science. 357 (1), 88-94 (2011).</li><li>Glatter, O., Scherf, G., Schillen, K., Brown, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Poly(ethylene+oxide)+Poly(propylene+oxide)+Triblock+Copolymer+(EO(27)-PO39-EO(27))+in+Aqueous-Solution.">Characterization of a Poly(ethylene oxide) Poly(propylene oxide) Triblock Copolymer (EO(27)-PO39-EO(27)) in Aqueous-Solution.</a> Macromolecules. 27 (21), 6046-6054 (1994).</li><li>Mittelbach, R., Glatter, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+structure+analysis+of+small-angle+scattering+data+from+polydisperse+colloidal+particles.">Direct structure analysis of small-angle scattering data from polydisperse colloidal particles.</a> Journal of Applied Crystallography. 31, 600-608 (1998).</li></ol>

没有利益冲突的声明。

比较程序的SWAXS系统的允许众多变量来确定实验分析。从分析中获得的参数，这些参数可以被用于不同的目的，根据样品和实验设置。 SAXS提供纳米尺度的对象的大小和形状有关下列内容的信息，而WAXS重点上的原子和微观尺度的结构（ 如分子晶格，晶胞尺寸对称性）。更具体地，在稀溶液中的颗粒，小角X射线散射可以研究回转，粒子大小和形状的半径;用于高密度的样品，小角X射线散射可?...

<table border="1"><tbody><tr><td> 产品名称 </td><td> 公司 </td></tr><tr><td>该系统3小型和广角X射线散射（SWAXS）相机</td><td> Hecus XRS和IBN， 奥地利格拉茨</td></tr><tr><td> GNOM </td><td> ATSAS 2.3包D. Svergun EMBL-汉堡</td></tr></tbody></table>

small and wide angle x-ray scattering studies of biological macromolecules in solution

在本文中，小型和广角X射线散射（SWAXS）分析大分子通过实验证明。 SWAXS是一种技术，其中X-射线被弹性散射在小角度（通常为0.1 - 5°）和宽角度（通常&gt; 5°）nm的范围中的由非均匀样品。这种技术提供的信息有关的形状，尺寸和分布的大分子，部分有序材料，孔径大小，和表面对体积之比的特征距离。小角度X射线散射（SAXS）是能够提供在1和200nm之间的大分子结构信息，而可以解决广角X射线散射（WAXS）更小布喇0.33 nm和0.49 nm的间距之间的样品的基础上具体的系统设置和检测器。的间距从布拉格定律确定，是依赖于波长和入射角。 在一个SWAXS实验，材料可以是固体或液体，并可能含有固体，液体或气体的域（所谓的粒子）的同一个或另一个的材料中的任何组合。 SWAXS的应用是非常广泛的，包括所有的类型:金属，复合材料，水泥，油，聚合物，塑料，蛋白质，食品和药品的胶体。对于固体样品的厚度被限制为约5毫米。 实验室为基础的SWAXS仪器的使用本文中详细介绍。可用的软件（ 例如 ，GNOM ATSAS 2.3包由D. Svergun的EMBL-的汉堡和EasySWAXS软件的）的SWAXS系统，可以进行的实验，以确定某些参数给定的样本。生物大分子实验的一个例子，是一种水基的水性缓冲液中，可以选择并通过许多方法制备的2％（重量）中溶菌酶的分析。样品制备遵循以下准则编制的示例部分。通过SWAXS实验，的溶菌酶的重要的结构参数， 例如 ，可以分析的回转半径。

小角X射线散射和WAXS完全可以提供结构信息的样品通过以下参数:回转，颗粒的大小和形状，解决方案的结构因子，具体的内表面和孔径大小，晶格的类型和尺寸，和电子密度1的半径。也可应用于小角X射线散射和WAXS研究蛋白质动力学2。 的结构信息SWAXS实验得到的实验检测到的光谱和该系统的计算结果进行比较。的计算结果，计算在一个合理的有效电位?...

Watch this Scientific Journal Video about Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution at JoVE.com

溶液中生物大分子的小广角X射线散射研究- 中国优秀硕士学位论文全文数据库生物工程|视频

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

小型和广角X-射线散射（SWAXS）过程的演示已经成为有助于在研究生物大分子。通过使用特定的角度的方法和准备的仪器和程序，从SWAXS的实验数据显示大分子的原子和纳米尺度的表征。

小型和广角X-射线散射研究生物大分子的解决方案

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

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18.1K 次观看.  Rensselaer Polytechnic Institute. 小型和广角X-射线散射（SWAXS）过程的演示已经成为有助于在研究生物大分子。通过使用特定的角度的方法和准备的仪器和程序，从SWAXS的实验数据显示大分子的原子和纳米尺度的表征。

视频: Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

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

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (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, ...

科研

生物化学

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

结合 x 射线晶体学与小角度 x 射线散射到模型的非结构化区域的 Nsa1 从s 酿酒酵母

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

用于膜蛋白结构分析和从头建模的小角中子散射实验中的对比匹配洗涤剂

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

小角度 x 射线散射溶液中大分子的结构研究

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

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. ...

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 ...

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). The present work describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

The present protocol describes methods for investigating the structure and dynamics of two model proteins that have an important role in human health. The technique combines bench-top biophysical characterization with neutron spin echo spectroscopy to access the dynamics at time and length scales relevant for protein interdomain motions.

通过中子自旋回声光谱研究蛋白质动力学

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The ...

工程学

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

通过微透析对芯片上的蛋白质进行结晶，用于 原位 X射线衍射研究

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

化学

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

在小角度中子衍射仪KWS-2研究软物质和生物系统在广泛的长度尺度从纳米，微米尺寸

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

生物工程

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

小型和广角X-射线散射研究生物大分子的解决方案

摘要

探索更多视频

此视频中的章节

在线体积排阻和离子交换色谱法SAXS光束线

结合 x 射线晶体学与小角度 x 射线散射到模型的非结构化区域的 Nsa1 从s 酿酒酵母

用于膜蛋白结构分析和从头建模的小角中子散射实验中的对比匹配洗涤剂

小角度 x 射线散射溶液中大分子的结构研究

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

通过 EFA 去卷和散射分析 SEC-SAXS 数据

通过中子自旋回声光谱研究蛋白质动力学

通过微透析对芯片上的蛋白质进行结晶，用于原位 X射线衍射研究

在小角度中子衍射仪KWS-2研究软物质和生物系统在广泛的长度尺度从纳米，微米尺寸

高分辨率中子光谱研究蛋白质和水合水的皮秒纳秒动力学

小型和广角X-射线散射研究生物大分子的解决方案

摘要

探索更多视频

此视频中的章节

在线体积排阻和离子交换色谱法SAXS光束线

结合 x 射线晶体学与小角度 x 射线散射到模型的非结构化区域的 Nsa1 从s 酿酒酵母

用于膜蛋白结构分析和从头建模的小角中子散射实验中的对比匹配洗涤剂

小角度 x 射线散射溶液中大分子的结构研究

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

通过 EFA 去卷和散射分析 SEC-SAXS 数据

通过中子自旋回声光谱研究蛋白质动力学

通过微透析对芯片上的蛋白质进行结晶，用于 原位 X射线衍射研究

在小角度中子衍射仪KWS-2研究软物质和生物系统在广泛的长度尺度从纳米，微米尺寸

高分辨率中子光谱研究蛋白质和水合水的皮秒纳秒动力学

通过微透析对芯片上的蛋白质进行结晶，用于原位 X射线衍射研究