这项工作是由BBSRC（BB/1006478/1）的部分资助。 PRR是英国皇家学会大学研究奖学金的获得者。

1。战略思考<ol><li>的籽晶用于microseeding实验的选择将取决于实验的目的而有所不同。在项目开始的时候是有帮助的找几个结晶命中，可以为水晶优化提供可供选择的出发点。 RMMS大大减少了对晶体优化，因为优质的晶体更容易在相图， 即在该地区，其中在干扰或核系统返回到平衡的稳区种植。因此，我们建议使用RMMS经常只要第一晶体获得（或者更准确的说，只要第一晶体停止生长）。 </li><li>对于首轮RMMS的，种子股票应尽可能多晶材料尽可能进行。如果只有一个孔是可用的包含晶体，或者如果晶粒是小的，也可能是有帮助的设置的多个重复原始命中（没有播种），以增加结晶的供给。但是，如果几个不同的检索结果得到，晶种可以从几个条件可以收获并混合在一起。 </li><li>为了避免相分离，在高盐条件下生长的晶体应分别从高PEG条件下生长的晶体收获。如果从多口井的结晶混合，贮存器的解决方案，是不容易得到的盐的晶体应选择种子悬浮液中。例如，应避免使用高浓度的磷酸盐，硫酸盐，钙和镁。 </li><li>后来在一个项目中，寻找具有不同的单元电池的结晶，以改善衍射，避免孪晶的晶体，得到晶体，适合在结合配体，它可能是很重要的。在该阶段只有最适合的晶体（ 如那些衍射最好）应该被用来使种子的库存。有时重复几轮microseeding都是必需的，其中仅“最佳“晶体是用来使种子股票的后续回合。如果可能的话，一个”中性“沉淀剂，如PEG 3000应该被用来在暂停晶种，以鼓励新的晶体接触和结晶复合物，可能是不稳定的高盐的解决方案12。 </li><li>经典microseeding实验中，在一个单一的结晶状态时，常常是有帮助以下的一个有希望的条件，并在随后的优化的鉴定。它往往是必要的稀释种子储备，以获得每一滴水晶体所需的号码。 “组合”microseeding实验（其中一系列的增加摊薄种子储备被添加到一个结晶条件）是一种快速的方法找到种子股票的最佳稀释。下面这种方法进行说明。 </li></ol> 2。种子股票的制备<ol><li>做一个圆形玻璃探头使用本生灯火焰玻璃巴斯德吸管。 <ol><li>加热靠近中间的吸管，直到它变得柔软，然后迅速从火焰中删除，并把它画出来了拉开两端。旨在拉动玻璃下降到直径小于0.25毫米。 </li><li>打破玻璃的地步，它是在0.25毫米和简要投身破碎端插入火焰。重复该步骤直至玻璃与形成于末直径为〜0.75毫米半球。这个探针是用于破碎晶体材料是有用的，因为它是适当的大小，以0.01-1.0毫米刻度撞击固态物体和它不损坏的结晶托盘子阱或盖玻片的塑料表面上。较大的探针（〜1.0毫米）可以更有效地粉碎小晶体，因为晶体的玻璃和塑料之间被困。 </li></ol></li><li>将装有冰种珠1.5 ml离心管中。 </li><li>使用双目显微镜或晶体成像系统检查预先建立结晶托盘，然后选择一个或多个APpropriate从托盘井从中收获的种子储备一代的晶体材料。任何材料都可以使用，包括细的针头，球晶，微晶和不规则，形成不良的结晶。该种子库应尽快之后晶体停止生长做，因为旧的材料更可能是部分交联，从而减少其在播种试验适用性。如有疑问形式存在于结晶材料可以收获是否是盐或蛋白质的井可以用前开口的UV荧光显微镜或成像系统的可视化，或者结晶性材料可以原位 X射线衍射分析来进行在托盘。当使用前一种方法的蛋白质晶体将紫外光照射下发出荧光，盐晶体将会出现无色。 </li><li>打开选定的结晶盘好。对于96孔坐在通过上胶密封片切割围绕降盘所选以及USIng的手术刀刀片。对于24孔悬滴盘使用镊子取出盖玻片。反转盖玻片，并将其放置在平稳的表面干净。去除50微升的储库溶液和该液体转移到含有种子珠确保管保持在冰上在整个离心管中。 </li><li>彻底粉碎了晶体中的subwell（96孔盘）或盖玻片（24孔托盘）下降，使用2.1节准备的玻璃探头，观察在显微镜下的结果。小晶体可能需要几分钟才能彻底粉碎。这个步骤允许实验者检查该晶体是易粉碎，因此不交联的，并且还认为它们不是盐晶体。盐晶体产生独特的点击，可以听到和感觉到他们被压碎时。如果晶体是坚韧，不易粉碎试图获得并使用新鲜的材料。 </li><li>删除5微升从种子珠管和t储液转拨到等效子阱（坐滴实验）或盖玻片（悬滴实验）含有结晶材料被收割。吸取该液体向上和向下的5-6倍，以悬浮尽可​​能多的子井或盖玻片的内容成为可能。返回暂停的种子珠管和重复此步骤两次以上，以确保尽可能多晶材料是收获的可能。 </li><li>涡流种子珠两分钟，停止每30秒冷却冰管。 </li><li>做系列稀释，由4至10在每个阶段的一个因素稀释种子库中贮存溶液。通常情况下，使用未稀释的种子的库存，当太多的结晶便可得到，因此使用稀释种子储备，以控制每孔的晶体的数目。在第一轮RMMS的，它不以稀释种子储备溶液是很重要的，因为更大的种子的浓度越结晶命中将获得的。 </li><li>如果它不被使用immediately，种子股票停牌，并稀释种子储备转移至-20℃，或-80℃冰箱保存。为了避免反复冻融周期，这可能会降低种子库的功效，这种材料存放在体积小等份， 即 10〜20微升。一旦冻结，种子储备可以保持下去，直到需要。 </li></ol> 3。建立结晶盘的<ol><li>根据不同的结晶设施和实验者的喜好的可用性，RMMS结晶检查可以使用任一24孔托盘采用悬滴气相扩散法，或96孔托盘采用坐滴气相扩散法进行。既可以建立手工结晶盘为RMMS，使用液体处理机器人的分配器，或者是两者的组合。 </li><li>一个典型的RMMS实验设置由专人将包括，1.0μL蛋白溶液，1.0微升结晶状态，0.5微升种子储备。一个典型的实验设置使用结晶机器人将包括，0.3μL蛋白溶液，0.2微升结晶条件，以及0.1微升种子储备。 </li><li>到在24孔悬滴盘执行RMMS筛选中，首先，使用手动移液管，转移300μl的每个条件的从在10ml单管格式的96孔结晶状态​​屏幕分成4×24孔pregreased结晶每个孔托盘。 <ol><li>转让1微升来自各300微升储各结晶状态到相应塑料盖玻片从正面和背面保护背衬条被去除的表面上。 </li><li>到1微升的每个结晶条件加1微升的蛋白质溶液，然后用0.5微升的晶种股票。反转每个盖玻片，使得液体的下降是朝下的和上面的适当的井的位置。 </li><li>按下向下的​​盖玻片，压缩seali纳克油脂，形成一个安全的密封。一旦所有96滴的建立，托盘应转移到孵化器或恒温室，其范围通常为4-18℃下储存。 </li></ol></li><li>在96孔坐滴盘首先传输20-50微升的每个条件的从在深井块格式的96孔结晶状态​​屏幕到结晶托盘的每个相应的井进行RMMS筛选。这个转印步骤可使用一个机器人结晶，或通过手动传输使用8道移液器进行。 <ol><li>转移储层，蛋白质和种子储备溶液至滴用手或用在3.1中详述的体积的机器人。一些市售的结晶机器人不接触的分配，以及使用这样的系统中进行种子库存转移可能会导致分注器或相关联的管道的阻塞。分配的顺序是可变的：一些机器人同时免除所有的解决方案。如果这是不可能的，首先免除的蛋白质，那么水库，然后种子储备。 </li><li>如果一个接触点胶机器人不可转让使用配有钝针的低容积的玻璃注射器的种子。通过传递它通过储液再分发种子股票进入每一滴冲洗针头。 </li><li>使用透明密封片密封托盘并转移到孵化器或恒温室进行存储。 </li></ol></li></ol> 4。结晶盘的检查<ol><li>想象使用一个双目显微镜或晶体成像系统中的所有结晶实验。前者通常能提供使用者更好地控制深度图和放大的程度大于后者，但更耗费时间。 </li><li>检查每个sub-well/coverslip顺序和记录晶体形成的任何证据。实验应检查一次，每24小时5天以下，并成立了Ñ​​随后连用7天为4周。 </li><li>经常需要RMMS的多次循环优化衍射级晶体产生之前。 </li></ol>

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我们什么都没有透露。

在本文中，我们描述了RMMS蛋白质结晶筛选的一般方法。使用两个测试蛋白质一个显著增强的结晶成功率用这种方法我们已经证明。使用使用RMMS和非RMMS方法产生的结晶的一个子集的同步加速器辐射衍射分析表明使用任一方法生长，虽然以前的作者已报道，良好品质的晶体更容易在RMMS实验11生长晶体间偏差小衍射质量， 13。 HEWL和背光补偿可能给非典型的结果，因为他们特别容易结?...

由佩鲁茨，肯德鲁和同事在确定血红蛋白和肌红蛋白的结构，在结构基因组学财团的现代高通量自动化流水线及其初步应用，大分子X射线晶体学已为我们提供了前所未有的结构性一窥世界的蛋白质。这种技术仍然是最广泛应用的实验方法，允许蛋白质结构的直接可视化在原子，或接近原子分辨率（ 即在1-3的范围内）。一个先决条件被应用到的蛋白质X-射线衍射是，它必须首先被结晶化，这是此阶段中残留的结构确定的一个最大限速步骤由衍射方法1，2的过程。尽管在我们的理解蛋白质结晶的方法，并且在结晶画面的质量和可用性的重大改进显著进步，托盘，以及相关的技术，但它仍然无法可靠地预测结晶成功3的可能性。生物化学和生物物理方法可用于评估是否感兴趣显示器的蛋白质晶体成核和生长良好的特性， 即它是良好的折叠，均匀的，单分散性等，然而，在没有办法这些见解提供结晶的最终的预测倾向。 播种长期以来一直声称是用于改善的数目，大小，和现有的晶体或结晶材料4-7的质量的可行方法。这种方法是基于的前提是，支持晶体成核的条件可能不是最优的用于随后的晶体生长，反之亦然。通过将核物质从一种状态到另一种，人们可能试图有效地分离这些过程，以新的，迄今未开发的结晶太空，使访问，并因此增加在筛选试验的总体成功率。建立的方法已被记录为（ⅰ）macroseeding，其全部的单晶从一个状态转移到另一个8，（ⅱ）条纹播种，核材料的转移，通常由定向的压力，例如，用应用程序得到猫的晶须到现有的晶体，然后通过随后的晶须的通路通过一个新的结晶降9，以及（iii）“经典的”microseeding的表面的晶体“种子”，以收获所产生的传输结晶粉碎物（或结晶性材料），到类似的，产生的种子10的条件。值得注意的是所有这三种方法都是费时和可扩展性很差，当然在比较什么是可以实现的现代化液体处理机器人技术的结晶。这些因素促成，在一定程度上，至少，在人们认为种子ING是一种方法，只访问时，其他方法都未能见效。 随机矩阵microseeding（RMMS）是最近的创新方法，结合了传统microseeding与高通量筛选和可扩展性11-13的好处。这种方法依赖于生成器的种子的库存，从核的结晶材料，其可以在一个标准的96条件结晶屏等分入/到每个sub-well/coverslip生产。此方法适用于两个坐或悬滴蒸气扩散实验中，通过手或使用液体处理机器人，在24孔或96孔盘格式建立。 RMMS已经通过实验证实，以显著提高结晶的成功率，并产生更大的衍射质量和数量11，13，14的晶体，并代表了晶体学家“在邻方法阿森纳一种创新的工具ngoing对结晶成功的努力。在这里，我们描述了用于RMMS的一般方法，并提供采样数据表示该技术的有效性。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>MRC 96 well crystallization trays</td>
			<td>Molecular Dimensions Ltd</td>
			<td>MD11-00-100</td>
			<td>Non-UV compatible, for screens established by robot</td>
		</tr>
		<tr>
			<td>ClearView sealing sheets</td>
			<td>Molecular Dimensions Ltd</td>
			<td>MD6-01S</td>
			<td></td>
		</tr>
		<tr>
			<td>Hen egg white lyzozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td>~95% purity</td>
		</tr>
		<tr>
			<td>Bovine liver catylase</td>
			<td>Sigma-Aldrich</td>
			<td>C9322</td>
			<td>&gt;95% purity</td>
		</tr>
		<tr>
			<td>Xylanase</td>
			<td>Hampton Research</td>
			<td>HR7-104</td>
			<td></td>
		</tr>
		<tr>
			<td>Thaumatin from Thaumatococcus daniellii</td>
			<td>Sigma-Aldrich</td>
			<td>T7630</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolysin from Bacillus thermoproteolyticus rokko</td>
			<td>Sigma-Aldrich</td>
			<td>P1512</td>
			<td></td>
		</tr>
		<tr>
			<td>JCSG-plus HT-96 screen</td>
			<td>Molecular Dimensions Ltd</td>
			<td>MD1-40</td>
			<td>For screens established by robot</td>
		</tr>
		<tr>
			<td>PACT premier HT-96 screen</td>
			<td>Molecular Dimensions Ltd</td>
			<td>MD1-36</td>
			<td>For screens established by robot</td>
		</tr>
		<tr>
			<td>Morpheus HT-96 screen</td>
			<td>Molecular Dimensions Ltd</td>
			<td>MD1-47</td>
			<td>For screens established by robot</td>
		</tr>
		<tr>
			<td>Crystal Phoenix liquid handling system</td>
			<td>Art Robbins Instruments</td>
			<td>602-0001-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Seed bead kit</td>
			<td>Hampton Research</td>
			<td>HR2-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular stereo microscope</td>
			<td>Leica</td>
			<td>M165C</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Sigma-Aldrich</td>
			<td>S2646-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>ErgoOne 0.1-2.5 &mu;l pipette</td>
			<td>Starlab</td>
			<td>S7100-0125</td>
			<td></td>
		</tr>
		<tr>
			<td>ErgoOne 2-20 &mu;l pipette</td>
			<td>Starlab</td>
			<td>S7100-0221</td>
			<td></td>
		</tr>
		<tr>
			<td>ErgoOne 100-1000 &mu;l pipette</td>
			<td>Starlab</td>
			<td>S7100-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>JCSG-plus screen</td>
			<td>Molecular Dimensions Ltd</td>
			<td>MD1-37</td>
			<td>For screens established by hand</td>
		</tr>
		<tr>
			<td>PACT premier screen</td>
			<td>Molecular Dimensions Ltd</td>
			<td>MD1-29</td>
			<td>For screens established by hand</td>
		</tr>
		<tr>
			<td>Morpheus screen</td>
			<td>Molecular Dimensions Ltd</td>
			<td>MD1-46</td>
			<td>For screens established by hand</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Sigma-Aldrich</td>
			<td>T5415-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>CrystalClene coverslips 18 mm</td>
			<td>Molecular Dimensions Ltd</td>
			<td>MD4-17</td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml glass Pasteur pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>Z722669</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>02-215-360</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well XRL crystallization tray</td>
			<td>Molecular Dimensions Limited</td>
			<td>MD3-11</td>
			<td>For screens established by hand</td>
		</tr>
		<tr>
			<td>30% (w/v) PEG 8000, 0.2 M ammonium sulfate, 0.1 M sodium cacodylate pH 6.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20% (w/v) PEG 8000, 0.2 M magnesium acetate, 0.1 M sodium cacodylate pH 6.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20% (w/v) PEG 6000, 100 mM citric acid pH 5.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

improving the success rate of protein crystallization by random microseed matrix screening

随机microseed基质筛选（RMMS）是在其中晶种加入到随机屏幕的蛋白质结晶技术。通过增加晶体蛋白质的相图介稳区将增长的可能性，额外的结晶导线通常获得的，​​所产生的晶体的质量可能会增加，并提供晶体数据收集和浸泡实验的良好供应。这里我们描述了可应用于任一坐滴或悬滴蒸气扩散实验中，通过手或使用液体处理机器人，在96孔或24孔盘格式建立RMMS的一般方法。

一个RMMS实验（A）示例以证明RMMS筛查的有效性，我们应用此方法到的鸡蛋白溶菌酶（HEWL）和牛肝脏过氧化氢酶（BLC）的结晶。这两个酶是突出的结晶和结构上良好表征的目标15，16。这样既提供出色的考试科目，用以说明增强结晶成功率可达到与RMMS。结晶实验是建立于96孔用液体处理机器人坐滴盘的格式。 HEWL和BLC的溶液通过分别在20mM的Tris-HCl，150mM的氯化钠，pH值7.5，?...

Watch this Scientific Journal Video about Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening at JoVE.com

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

在这里，我们描述了随机microseed矩阵筛选的一般方法。这种技术被示为显著增加的蛋白质结晶筛选实验的成功率，减少需要优化，并提供晶体的数据收集和配体浸泡实验的可靠供应。

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 ...

improving-success-rate-protein-crystallization-random-microseed

Research

JoVE Journal

Biology

17.7K 次观看.  University of Bristol. 在这里，我们描述了随机microseed矩阵筛选的一般方法。这种技术被示为显著增加的蛋白质结晶筛选实验的成功率，减少需要优化，并提供晶体的数据收集和配体浸泡实验的可靠供应。 

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

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

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

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 ...

科研

生物学

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

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 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 ...

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 ...

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

酶谱法检测功能的基质金属蛋白酶

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

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 ...

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

单粒子电子显微镜外切体综合重建，使用随机的锥形倾斜方法

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

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 ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

MBP基于标记策略的Mgm101重组蛋白的制备

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...