Name: improving the success rate of protein crystallization by random microseed matrix screening
Uploaded: 2013-08-31T10:00:00
Description: Watch this Scientific Journal Video about Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening at JoVE.com

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）。 </...

<ol>
	<li>Bergfors, T. <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.">Protein Crystallization.</a> IUL Biotechnology Series. , (2009).</li><li>Rupp, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomolecular Crystallography: Priciples, Practice and Application to Structural Biology. , (2010).</li><li>Babnigg, G., Joachimiak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+protein+crystallization+propensity+from+protein+sequence.">Predicting protein crystallization propensity from protein sequence.</a> J. Struct. Funct. Genomics. 11 (1), 71-80 (2010).</li><li>Bergfors, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeds+to+crystals.">Seeds to crystals.</a> J. Struct. Biol. 142 (1), 66-76 (2003).</li><li>Ireton, G. C., Stoddard, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microseed+matrix+screening+to+improve+crystals+of+yeast+cytosine+deaminase.">Microseed matrix screening to improve crystals of yeast cytosine deaminase.</a> Acta. Crystallogr. D. Biol. Crystallogr. 60, 601-605 (2004).</li><li>Zhu, D. Y., Zhu, Y. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+protein+crystal+growth+through+dynamic+seeding.">Optimizing protein crystal growth through dynamic seeding.</a> Acta. Crystallogr. D. Biol. Crystallogr. 61 (Pt 6), 772-775 (2005).</li><li>Bergfors, T. <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+methods+for+nonautomated+crystallization+laboratories.">Screening and optimization methods for nonautomated crystallization laboratories.</a> Methods Mol. Biol. 363, 131-151 (2007).</li><li>Xu, L., Butcher, S. J., et al. <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+analysis+of+receptor-binding+protein+P2+of+bacteriophage+PRD1.">Crystallization and preliminary X-ray analysis of receptor-binding protein P2 of bacteriophage PRD1.</a> J. Struct. Biol. 131 (2), 159-1563 (2000).</li><li>Rangarajan, E. S., Izard, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+diffraction+of+full-length+human+selenomethionyl+metavinculin+crystals+by+streak-seeding.">Improving the diffraction of full-length human selenomethionyl metavinculin crystals by streak-seeding.</a> Acta. Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 66 (Pt 12), 1617-1620 (2010).</li><li>Kadirvelraj, R., Harris, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+optimization+of+crystals+of+rhamnogalacturonan+lyase+from+Aspergillus+aculeatus.">A stepwise optimization of crystals of rhamnogalacturonan lyase from Aspergillus aculeatus.</a> Acta. Crystallogr. D. Biol . Crystallogr. 58 (Pt 8), 1346-1349 (2002).</li><li>D'Arcy, A., Villard, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+microseed+matrix-screening+method+for+protein+crystallization.">An automated microseed matrix-screening method for protein crystallization.</a> Acta. Crystallogr. D. Biol. Crystallogr. 63 (Pt 4), 550-554 (2007).</li><li>Shaw Stewart, P. D., Kolek, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+Microseeding:+A+Theoretical+and+Practical+Exploration+of+Seed+Stability+and+Seeding+Techniques+for+Successful+Protein+Crystallization.">Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization.</a> Crystal Growth &amp; Design. 11 (8), 3432-3441 (2011).</li><li>Obmolova, G., Malia, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoting+crystallization+of+antibody-antigen+complexes+via+microseed+matrix+screening.">Promoting crystallization of antibody-antigen complexes via microseed matrix screening.</a> Acta. Crystallogr. D. Biol. Crystallogr. 66, 927-933 (2010).</li><li>Villasenor, A. G., Wong, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+matrix+microseeding:+improving+protein+crystal+growth+with+minimal+chemical+bias.">Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias.</a> Acta Crystallogr D Biol Crystallogr. 66 (Pt 5), 568-5676 (2010).</li><li>Strynadka, N. C., James, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysozyme:+a+model+enzyme+in+protein+crystallography.">Lysozyme: a model enzyme in protein crystallography.</a> EXS. 75, 185-222 (1996).</li><li>Diaz, A., Loewen, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thirty+years+of+heme+catalases+structural+biology.">Thirty years of heme catalases structural biology.</a> Arch. Biochem. 525 (2), 102-110 (2012).</li></ol>

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 ...

Research

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Biology

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 ...

科研

生物学

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 ...

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 ...

Detection of Functional Matrix Metalloproteinases by Zymography

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

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 ...

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

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

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

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

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 ...

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 ...