Name: assessing two-dimensional crystallization trials of small membrane proteins for structural biology studies by electron crystallography
Uploaded: 2010-10-29T17:00:00
Description: Watch this Scientific Journal Video about Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography at JoVE.com

我们感谢我们的合作者提供了宝贵的蛋白质样品，这有助于我们的方法相关的经验和观察一些。德博Schmalzing好心提供了机会，为fr加入这个项目。芭芭拉Armbruster，雅各Brink和德里克米尔斯是他们的杰出的帮助和设备上的投入表示感谢。经费是由美国国立卫生研究院授予HL090630。

1。电网制备二维结晶试验<ol><li>碳包400目铜EM电网负染色准备。醋酸铀是经常使用的，并提供在存储解决方案方面的长期染色前使用，以及适合长期存储网格的几个月。相比之下，其他负面的污渍，如铀酸盐，同时提供优良的染色，需要新鲜的。对于大量的二维结晶试验筛选用于电网的快速准备，负染的修改后的版本使用。 2μL的样品量吸管将碳覆盖EM网格和孵育60秒。这是一个撕成片的Whatman＃4...

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	<li>Johansen, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+field+electron+microscopy+of+biological+specimens+V.+A+low+dose+pre-irradiation+procedure+reducing+beam+damage.">Bright field electron microscopy of biological specimens V. A low dose pre-irradiation procedure reducing beam damage.</a> Micron. 7, 145-156 (1976).</li><li>Schmidt-Krey, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+crystallography+of+membrane+proteins:+Two-dimensional+crystallization+and+screening+by+electron+microscopy.">Electron crystallography of membrane proteins: Two-dimensional crystallization and screening by electron microscopy.</a> Methods. 41, 417-426 (2007).</li><li>Wang, D. N., K&#252;hlbrandt, W. <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+electron+crystallography+of+light-harvesting+chlorophyll+a/b-protein+complex+in+three+different+media.">High-resolution electron crystallography of light-harvesting chlorophyll a/b-protein complex in three different media.</a> J Mol Biol. 217, 691-699 (1991).</li><li>Schmidt-Krey, I., Rubinstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryomicroscopy+of+membrane+proteins:+specimen+preparation+for+two-dimensional+crystals+and+single+particles.">Electron cryomicroscopy of membrane proteins: specimen preparation for two-dimensional crystals and single particles.</a> Micron. , (2010).</li><li>Schmidt-Krey, I., Mutucumarana, V., Haase, W., Stafford, D. W., K&#252;hlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+of+human+vitamin+K-dependent+&amp;gamma;-glutamyl+carboxylase.">Two-dimensional crystallization of human vitamin K-dependent &amp;gamma;-glutamyl carboxylase.</a> J Struct Biol. 157, 437-442 (2007).</li><li>Trachtenberg, S., DeRosier, D. J., Zemlin, F., Beckmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-helical+perturbations+of+the+flagellar+filament:+Salmonella+typhimurium+SJW117+at+9.6+&amp;Aring;+resolution.">Non-helical perturbations of the flagellar filament: Salmonella typhimurium SJW117 at 9.6 &amp;Aring; resolution.</a> J Mol Biol. 276, 759-773 (1998).</li><li>Zhao, G., Johnson, M. C., Schnell, J. R., Kanaoka, Y., Irikura, D., Lam, B. K., Austen, K. F., Schmidt-Krey, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+conditions+of+human+leukotriene+C4+synthase+requiring+a+particularly+large+combination+of+specific+parameters.">Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring a particularly large combination of specific parameters.</a> J Struct Biol. 169, 450-454 (2010).</li><li>Cheng, A., Leung, A., Fellmann, D., Quispe, J., Suloway, C., Pulokas, J., Abeyrathne, P. D., Lam, J. S., Carragher, B., Potter, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+automated+screening+of+two-dimensional+crystals.">Towards automated screening of two-dimensional crystals.</a> J Struct Biol. 160, 324-331 (2007).</li><li>Vink, M., Derr, K. D., Love, J., Stokes, D. L., Ubarretxena-Belandia, 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+high-throughput+strategy+to+screen+2D+crystallization+trials+of+membrane+proteins.">A high-throughput strategy to screen 2D crystallization trials of membrane proteins.</a> J Struct Biol. 160, 295-304 (2007).</li></ol>

样品进行适当的评价，需要认真评估的足够数量的膜。例如，低至2％的晶体阵列成像proteoliposomes超过180的样品给了二维结晶条件的快速优化的关键信息。 蛋白质沉淀发生时，一个网格可能被放弃进一步的筛选后，在低倍率的检查，虽然偶尔出现部分沉淀的蛋白质。即使很小小于100 nm的膜虽然可以给有关订单的有用信息，并增加二维晶体大小的参数，可以在以下透析实验实施?...

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<td>400-mesh copper TEM grids coated with carbon film</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>forceps: regular and anti-capillary </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dumont #5 and Dumont N5AC or similar</td>
</tr>
<tr>
<td>Micropipette and pipette tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Whatman #4 filter paper</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1% uranyl acetate</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dialysis sample to be screened for 2D crystals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Glycerol/sucrose-free dialysis buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Optional</td>
</tr>
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<td>JEOL-1400 transmission electron microscope (TEM)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>similar 80 &#x2013; 120kV TEM equipped with an Lab6 or tungsten filament and film and/or CCD cameras (Gatan Orius SC1000 and/or UltraScan1000 CCD cameras and Gatan Digitial Micrograph software package or Tietz cameras (TVIPS)) </td>
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assessing two-dimensional crystallization trials of small membrane proteins for structural biology studies by electron crystallography

电子晶体学已经发展成为一个可以交替使用，也可以结合三维结晶和X射线晶体学研究膜蛋白的结构与功能的问题，以及可溶性蛋白的方法。二维（2D）晶体透射电子显微镜（EM）的筛查是在寻找关键的一步，优化，并选择冷冻电镜的高分辨率数据采集的样品。在这里，我们描述了大型和排序，以及小型二维数组，可以潜在的结晶条件的优化提供重要的信息在确定的基本步骤。 通过不同放大倍数的EM，在一系列关键参数的获取数据。下放大倍率上的形态和膜大小提供了宝贵的数据。在更高的放大倍率，确定可能的秩序和二维晶体的尺寸。在这种情况下，它是描述CCD相机和在线傅立叶变换是如何在更高的放大倍率用于评估顺序和大小proteoliposomes。 虽然膜蛋白的二维晶体，​​是最常用的透析重建的速度增长，筛选技术也同样适用于单层，帮助本地的二维晶体生产晶体，并下令可溶性蛋白质阵列。此外，这里介绍的方法适用于二维晶体更小，以及较大的膜蛋白，其中较小的蛋白质需要在识别相同数量的护理我们的例子中，较大的蛋白质晶格的筛选，可能会更容易识别在早期阶段的筛选。

Watch this Scientific Journal Video about Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography at JoVE.com

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

形成有序的膜蛋白质阵列的二维（2D）的结晶试验评价是一个非常关键的电子晶体而艰巨的任务。在这里，我们描述了我们的方法，筛选，并确定在15范围内的主要是小膜蛋白的二维晶体 - 90kDa。

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 ...

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Microcontact Printing of Proteins for Cell Biology

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The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

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Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Protein Crystallization for X-ray Crystallography

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 ...

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

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

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

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An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

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Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

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Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...