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滤纸（图1，视频）从边缘杂交，然后2μL1％醋酸铀立即应用，这是再次涂抹后，30日从电网的边缘第在谈到与滤纸撕边的边缘电​​网确保不拆除的proteoliposomes最佳去除液体。此外，在电网的边缘干燥，确保改进的碳薄膜的保存。必须小心，在准备和处理电网，微妙的碳薄膜的破损防止样品的附着力，并可能导致不准确的代表性样品。虽然较大的样本量5μL传统和常规电网准备，可节省宝贵的样品量减少2μL或更少2。 </li><li>为了产生尽可能大的膜，我们的一些样品需要透析缓冲甘油或蔗糖浓度高（10-20％）。这可能对电网准备的负面影响，如甘油或蔗糖是高度粘性和正确地渗透到缓冲区，因而不完全染色膜，防止醋酸铀。因此，一个可能的晶格会被遮蔽。可以通过离心的样品和替代甘油/蔗糖缓冲液（未显示），或电网交换缓冲区要么可水洗负染色类似与前几个周期的缓冲区或甘油/蔗糖在一个无缓冲3,4冷冻电镜采用的技术。 </li></ol> 2。评估EM二维结晶试验<ol><li>根据样品架类型，加载一个或多个网格。 2 - 10K，这将在这里被称为中间放大倍率的放大倍率，可以平均分配和膜，形态和大小，这是在实验室笔记本5注意到的分散程度的第一印象。适宜地区作为与CCD相机的帮助下代表概述或记录，如果CCD相机是不可用在胶片上，。 </li><li>概述整个样本/电网需要时，使用低倍率范围在大约400 - 800X倍。虽然不是每个网格就业，低倍率提供有价值的信息的帮助，在电网准备在几个方面的评价:负染色和潜在的碳薄膜，样品的浓度，对电网和可能proteoliposome分布不均的局部破损。用双筒望远镜或CCD摄像头，可能被视为个人方格。随着一些新兴市场有可能保存特别感兴趣的职位，放大倍率越高，可稍后再检查召回。 </li><li>一旦利益的网格区域已确定或低中间放大倍率，放大倍率约50K - 60K。根据膜，水晶和单位细胞​​的大小，如果知道的话，低为30K和高达80K高放大倍率的使用。放大倍率范围为30 - 80K将被称为高倍率筛选二维晶体的目的。聚焦发生在低剂量的设置焦点设置，图像/照片设定，或在感兴趣的区域附近，随后开关。 <ol class="lalpha"><li>在感兴趣的领域是否确实是一种膜含糊不清的情况下，检查样品的碳薄膜proteoliposomes，云母，或其他文物的大小。为此，边缘显示了典型的折叠和形态。 </li><li>现在感兴趣的领域进行检查与CCD相机。 CCD图像收集在30K - 80K放大，根据膜的大小，蛋白质或单位细胞大小，或称为结晶区​​。不一定是较小的和/或主要是疏水的膜蛋白的晶格可见CCD图像本身的视觉评估。整个图像要么是用于在线傅立叶变换（FT，或快速傅立叶变换FFT）。这个金融时报"将包含大量的噪音，但是，如果有序阵列。因此，减少盒装图像的大小，将允许改进的信号噪声比一个SMaL公司LER晶体，并便于识别。为此，框移到图像和现场的FT评估。 梁的强度/亮度调整，保持低剂量条件下的样品，以及心中的CCD相机设置。根据所使用的CCD摄像头，现场的FT的伽玛调整为有序阵列的最佳识别。过高的值可以掩盖斑点噪声贡献，和过低的伽玛值将防止从弱点被确定。这些较弱的斑点可能是由于较小的晶体阵列斑点勉强以上的噪音水平的FT。 虽然在更高的分辨率数据已经收集到少量的样品6，常用的负染的二维晶体的分辨率是有限的或不应该预计比大约15A的分辨率更好。一个散焦约-400纳米，不超过1-3点的订单，预计将很容易识别。样品一般都没有评估决议，冷冻电镜收集数据，将给予最高可实现分辨率的适当指示。指出虽然点和可能mosaicity的清晰度。 </li></ol></li><li>膜，以及不同的膜形貌，是为了在高放大倍率的评估。这是关键，特别是在开始或中间阶段的二维结晶试验，小而不是更大的proteoliposomes或膜补丁可以包含最有前途的领域。二维晶体非常低的百分比，将需要图像采集和FT，或大量的图像有序阵列的初步鉴定，因为经常会导致2,4,7的规模和质量的迅速改善光学衍射。 </li></ol> 3。代表性的成果理想的情况下下令proteoliposomes显示容易辨认，清晰的斑点。在线FT CCD图像或光学显微衍射大型和有序的晶体很容易识别。 的例子显示，在几微米大小的18kDa的小膜蛋白的二维晶体。 "金融时报"上的斑点很容易识别和尖锐。活英尺箱的运动表明，晶格是连续的，没有mosaicity。可确定一个更大的蛋白质与更广泛的水溶性域的晶格上的小屏幕的EM。 CCD图像采集和FT是要提供一个更好的评估手段和获得信息，例如，可能mosaicity（显示英尺）。当计算一个没有下令proteoliposome FT噪音，可以初步被误为斑点。虽然生活- FT框移动，然而，斑点就会消失。另一方面，小数组，将可疑的结晶，有其时现场FT是移动的图像区域，甚至略有剩余的固定点。此外，这些小晶体可以被识别，通常具有相同的晶胞尺寸，并在不同的FTS点之间的距离可以测量各种方式，如与一个特定大小的的圆圈。脂质体显示不同的晶格形态和FT。 在初步试验中遇到的沉淀并不少见。这里没有重组蛋白沉淀，需要从虽然小脂质聚集区分开来。样品出现沉淀在低倍率频繁转出脂质聚集在更高的放大倍率观察。在30 - 50K的检查后，这些黑暗结构的边缘，显示他们没有沉淀的蛋白质组成的膜。这些都是重要的观察脂质聚集的大小可能会增加，在下面的实验大膜。 评估样品的效果差，有时连接到一个低的膜的浓度，以防止正确和快速筛选。这常常被克服二维结晶的使用提出了更高的蛋白浓度的透析。另外，膜可以留几天在底部的Eppendorf管中，在储存过程中解决。在某些情况下发生膜迅速，或几乎即时解决，移液管底部，将导致对电网的膜密度较高。另一种更快的选项离心随后从试管底部采样的样本（1-3分钟，在3000-8000 RPM）。 在最佳条件下的样品将包含一个二维晶体的很大比例。这是没有必要的膜齐亮相目标，作为最大的和最有序的二维晶体选定的数据可视化的集合。这些类型的样品，将很容易确认时的结晶试验以及用于样品时重复冷冻电镜收集数据，从而导致在高分辨率图像的最大数目。 <img src="/files/ftp_upload/1846/1846fig1.jpg" alt="图1" /> 图1此图显示了一个撕成片的Whatman＃4滤纸杂交电网的边缘。

<ol>
	<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。 实验室在筛选8,9的过程中引入不同程度的有前途的自动化过程。然而，样品将仍然需要评价和知识的二维晶体的大小，形态和秩序的步骤。在未来的日子里，它可能会变得越来越小的二维晶体分析数据，以更高的分辨率，使得这些筛选步骤，更小，更关键的结晶领域更困难的评价。

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		<th>Material Name</th>
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<td>400-mesh copper TEM grids coated with carbon film</td>
<td>&nbsp;</td>
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<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>
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<td>Micropipette and pipette tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<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>
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<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>
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<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 ...

assessing-two-dimensional-crystallization-trials-small-membrane

Nanyang Technological University

Research

JoVE Journal

Biology

11.2K 次观看.  Georgia Institute of Technology. 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。

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

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

微接触印刷的蛋白细胞生物学

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

科研

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in 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

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by 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

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

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

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

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 ...

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

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

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

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

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

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

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

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

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

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

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

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

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 ...