Name: protein crystallization for x-ray crystallography
Uploaded: 2011-01-16T16:03:00
Description: Watch this Scientific Journal Video about Protein Crystallization for X-ray Crystallography at JoVE.com

这项工作是由宝来惠康研究者奖，以YM和布朗Coxe从美国耶鲁大学博士后奖学金到MD的支持。

材料： <ul><li>蛋白质样品 - 溶菌酶（50毫克/毫升） </li><li>悬滴24井托盘</li><li>坐滴24井托盘</li><li> Microbatch结晶96以及托盘</li><li>结晶解决方案（无论是商业用或自制） </li><li>硅脂</li><li> 5 mL注射器无鲁尔锁</li><li>硅化盖幻灯片</li><li>光封箱胶带</li><li>石蜡</li><li> 0.1-2微升微量低保留提示</li><li>镊子</li><li>专业湿巾</li></ul> 1。悬/坐在DROP PROCEDURE： <ol><li>对于一个初始画面，可以?...

<ol>
	<li>Bergfors, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein crystallization : techniques, strategies, and tips : a laboratory manual. , (1999).</li><li>Carter, C. W., Carter, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystallization+using+incomplete+factorial+experiments.">Protein crystallization using incomplete factorial experiments.</a> J Biol Chem. 254, 12219-12223 (1979).</li><li>Cudney, R., Patel, S., Weisgraber, K., Newhouse, Y., McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+optimization+strategies+for+macromolecular+crystal+growth.">Screening and optimization strategies for macromolecular crystal growth.</a> Acta Crystallogr D Biol Crystallogr. 50, 414-423 (1994).</li><li>Dessau, M., Chamovitz, D. A., Hirsch, 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=Expression,+purification+and+crystallization+of+a+PCI+domain+from+the+COP9+signalosome+subunit+7+(CSN7).">Expression, purification and crystallization of a PCI domain from the COP9 signalosome subunit 7 (CSN7).</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 62, 1138-1140 (2006).</li><li>Gilliland, G. L., Tung, M., Blakeslee, D. M., Ladner, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Macromolecule+Crystallization+Database,+Version+3.0:+new+features,+data+and+the+NASA+archive+for+protein+crystal+growth+data.">Biological Macromolecule Crystallization Database, Version 3.0: new features, data and the NASA archive for protein crystal growth data.</a> Acta Crystallogr D Biol Crystallogr. 50, 408-413 (1994).</li><li>Jancarik, J., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparse+matrix+sampling:+a+screening+method+for+crystallization+of+proteins.">Sparse matrix sampling: a screening method for crystallization of proteins.</a> J Appl Cryst. 23, 409-411 (1991).</li><li>Jancarik, J., Scott, W. G., Milligan, D. L., Koshland, D. E., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+study+of+the+ligand-binding+domain+of+the+bacterial+chemotaxis-mediating+aspartate+receptor+of+Salmonella+typhimurium.">Crystallization and preliminary X-ray diffraction study of the ligand-binding domain of the bacterial chemotaxis-mediating aspartate receptor of Salmonella typhimurium.</a> J Mol Biol. 221, 31-34 (1991).</li><li>Kimber, M. S., Vallee, F., Houston, S., Necakov, A., Skarina, T., Evdokimova, E., Beasley, S., Christendat, D., Savchenko, A., Arrowsmith, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+mining+crystallization+databases:+knowledge-based+approaches+to+optimize+protein+crystal+screens.">Data mining crystallization databases: knowledge-based approaches to optimize protein crystal screens.</a> Proteins. 51, 562-568 (2003).</li><li>Lorber, B., Sauter, C., Theobald-Dietrich, A., Moreno, A., Schellenberger, P., Robert, M. C., Capelle, B., Sanglier, S., Potier, N., Giege, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+growth+of+proteins,+nucleic+acids,+and+viruses+in+gels.">Crystal growth of proteins, nucleic acids, and viruses in gels.</a> Prog Biophys Mol Biol. 101, 13-25 (2009).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Crystallization of biological macromolecules. , (1999).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+protein+crystallization.">Introduction to protein crystallization.</a> Methods. 34, 254-265 (2004).</li><li>Page, R., Grzechnik, S. K., Canaves, J. M., Spraggon, G., Kreusch, A., Kuhn, P., Stevens, R. C., Lesley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+crystallization+strategy+for+structural+genomics:+an+optimized+two-tiered+crystallization+screen+against+the+Thermotoga+maritima+proteome.">Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome.</a> Acta Crystallogr D Biol Crystallogr. 59, 1028-1037 (2003).</li></ol>

在这篇文章中我们将介绍和演示蛋白质结晶当前的协议。自一个多步骤的过程中有几个注意事项要注意的一个需要。当工作与极少量（0.5-2μL）下拉由于蒸发干燥，是一个主要问题。因此，建议工作在良好的控制环境低的空气流动性，高湿度和严格的温度控制，并采取了一种技术，最大限度地减少暴露露天下降。出于这个原因，它运行在一个组织良好的的工作环境是必不可少的。所有试剂应准备?...

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<td>Lysozyme</td>
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<td>24 well VDX Plate</td>
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<td>24 well Cryschem Plate</td>
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<td>Dow Corning Vacuum Grease</td>
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<td>Siliconized glass circle coverslides</td>
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<td>100% paraffin oil</td>
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<td>Hampton research</td>
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<td>1.88 inch wide Crystal Clear Sealing Tape</td>
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<td>Hampton research</td>
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<td>96 Well Imp@ct Plate (Microbatch plate)</td>
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<td>Hampton research</td>
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protein crystallization for x-ray crystallography

使用生物大分子的三维结构来推断它们如何运作，是现代生物学的最重要领域之一。原子分辨率结构的可用性提供了一个深刻和独特的理解蛋白质的功能，并有助于揭示活细胞的内部工作。迄今为止，86％的蛋白质数据银行（RCSB PDB）项的确定，采用X射线晶体学的大分子结构。 为了获得适合晶体，晶体学研究，大分子（如蛋白质，核酸，蛋白质 - 蛋白质复合物或蛋白质 - 核酸复合物）必须得到净化同质化，或尽可能接近同质化。同质化的准备是获得晶体衍射高分辨率（Bergfors，1999年;麦弗逊，1999年）的一个关键因素。 到过饱和的高分子结晶需要。样本，因此，应集中到尽可能高的浓度，而不会造成聚集或沉淀的高分子（通常为2-50毫克/毫升）。样品引入沉淀剂，能促进蛋白质晶体的成核的解决方案，这可能会导致大从溶液中的三维晶体生长。获得晶体主要有两种技术：气相扩散和间歇结晶。一滴含有沉淀和蛋白质溶液的混合物蒸气扩散，是密封在一个纯粹的沉淀室。水蒸汽，然后扩散的下降，​​直到渗透压下降，沉淀的是平等的（图1A）。脱水下降导致蛋白质和沉淀缓慢的浓度，直到实现平衡，坐落在相图的晶体核区。批处理的方法依赖于使混合蛋白的蛋白质直接进入核区与适量的沉淀（图1B）。这种方法通常是下一个石蜡/矿物油混合，以防止下拉水的扩散。 在这里，我们将演示两种蒸汽扩散实验装置，挂在下降，坐滴，除了批量下油的结晶。

Crystallization is usually referred to as the bottleneck of X-ray crystallography. A sparse matrix incomplete factorial screen of precipitating conditions typically produces many different types of protein aggregation and precipitation, among them large single crystals. If the protein or precipitant concentrations are too high one can see brown matter with no distinct shape and size (amorphous precipitation). When the solution is undersaturated, the drop will often be completely clear and devoid of any kind of precipitat...

Watch this Scientific Journal Video about Protein Crystallization for X-ray Crystallography at JoVE.com

Protein Crystallization for X-ray Crystallography

一个分子的三维结构的分子的功能提供了一个独特的理解。在近原子分辨率的结构测定的主要方法是X射线晶体学。在这里，我们展示了取得任何给定的高分子三维晶体，通过X射线晶体结构的决心适合的电流的方法。

X射线晶体学的蛋白质结晶

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Research

JoVE Journal

Biology

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

评估电子晶体结构生物学研究的小膜蛋白的二维结晶试验

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Crystallization of Membrane Proteins in Lipidic Mesophases

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

基础X射线计算机断层数据的三维打印设置

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

提高蛋白质结晶的成功率随机Microseed矩阵筛选

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...