Name: creating and applying a reference to facilitate the discussion and classification of proteins in a diverse group
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Description: Watch this Scientific Journal Video about Creating and Applying a Reference to Facilitate the Discussion and Classification of Proteins in a Diverse Group at JoVE.com

正在缓步动物抗菌肽的研究得到了校内资金来自美国中西部大学研究办公室和赞助程序 (ORSP)。ORSP 没有任何作用，研究设计、 数据收集、 分析、 解释或手稿的准备。

1.确定定义功能的蛋白质组的 <ol><li>咨询以前的出版物，以确定是否有必要考虑组的一部分的功能方面达成共识。注意到的任何不一致之处或意见研究团体之间的分歧，包括可能有助于区分另一个亚组的特点。</li>
	<li>如果以往文献不能解决最大特色，使用序列作为起点来识别保守的特点被认为集团代表。</li>
</ol> 2。收集有关序列 <ol><li>如果已编写审核，包括分析序列，所?...

<ol>
	<li>Matsuyama, K., Natori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Three+Antibacterial+Proteins+from+the+Culture+Medium+of+NIH-Sape-4,+an+Embryonic+Cell+Line+of+Sarcophaga+peregrina.">Purification of Three Antibacterial Proteins from the Culture Medium of NIH-Sape-4, an Embryonic Cell Line of Sarcophaga peregrina.</a> J Biol Chem. 263 (32), 17112-17116 (1988).</li><li>Lambert, 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=Insect+immunity:+Isolation+from+immune+blood+of+the+dipteran+Phormia+terranovae.+of+two+insect+antibacterial+peptides+with+sequence+homology+to+rabbit+lung+macrophage+bactericidal+peptides.">Insect immunity: Isolation from immune blood of the dipteran Phormia terranovae. of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bactericidal peptides.</a> PNAS. 86 (262-266), (1989).</li><li>Dimarcq, J. -. 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Comp. Immunol. 39, 79-84 (2013).</li><li>Bonmatin, J. -. M., 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=Two-dimensional+1H+NMR+study+of+recombinant+insect+defensin+A+in+water:+Resonance+assignments,+secondary+structure+and+global+folding.">Two-dimensional 1H NMR study of recombinant insect defensin A in water: Resonance assignments, secondary structure and global folding.</a> J Biomol NMR. 2 (3), 235-256 (1992).</li><li>Cornet, B., 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=Refined+three-dimensional+solution+structure+of+insect+defensin+A.">Refined three-dimensional solution structure of insect defensin A.</a> Structure. 3 (5), 435-448 (1995).</li><li>Murzin, A. G., Brenner, S. 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D., 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=Gene3D:+expanding+the+utility+of+domain+assignments.">Gene3D: expanding the utility of domain assignments.</a> Nucleic Acids Res. 44, 404-409 (2016).</li><li>Tarr, D. E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+a+reference+array+for+the+CS-ab+superfamily+of+defensive+peptides.">Establishing a reference array for the CS-ab superfamily of defensive peptides.</a> BMC Res Notes. 9, 490 (2016).</li><li>Shafee, T. M. A., Robinson, A. J., van der Weerden, N., Anderson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+homology+guided+alignment+of+cysteine+rich+proteins.">Structural homology guided alignment of cysteine rich proteins.</a> SpringerPlus. 5 (27), (2016).</li><li>Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Local+Alignment+Search+Tool.">Basic Local Alignment Search Tool.</a> J Mol Biol. 215 (3), 403-410 (1990).</li><li>Duckert, P., Brunak, S., Blom, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+proprotein+convertase+cleavage+sites.">Prediction of proprotein convertase cleavage sites.</a> Protein Eng Des Sel. 17 (1), 107-112 (2004).</li><li>Petersen, T. N., Brunak, S., von Heijne, G., Nielsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SignalP+4.0:discriminating+signal+peptides+from+transmembrane+regions.">SignalP 4.0:discriminating signal peptides from transmembrane regions.</a> Nat Methods. 8, 785-786 (2011).</li><li>Kobayashi, Y., 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=The+cysteine-stabilized+a-helix:+A+common+structural+motif+of+ion-channel+blocking+neurotoxic+peptides.">The cysteine-stabilized a-helix: A common structural motif of ion-channel blocking neurotoxic peptides.</a> Biopolymers. 31, 1213-1220 (1991).</li><li>Gao, B., del Carmen Rodriguez, M., Lanz-Mendoza, H., Zhu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AdDLP,+a+bacterial+defensin-like+peptide,+exhibits+anti-Plasmodium.+activity.">AdDLP, a bacterial defensin-like peptide, exhibits anti-Plasmodium. activity.</a> Biochem Biophys Res Commun. 387, 393-398 (2009).</li><li>Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEGA6:+Molecular+Evolutionary+Genetics+Analysis.">MEGA6: Molecular Evolutionary Genetics Analysis.</a> Mol Biol Evol. 30 (12), 2725-2729 (2013).</li><li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUSCLE:+multiple+sequence+alignment+with+high+accuracy+and+high+throughput.">MUSCLE: multiple sequence alignment with high accuracy and high throughput.</a> Nucleic Acids Res. 32 (5), 1792-1797 (2004).</li><li>Ronquist, F., Huelsenbeck, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MrBayes+3:+Bayesian+phylogenetic+inference+under+mixed+models.">MrBayes 3: Bayesian phylogenetic inference under mixed models.</a> Bioinformatics. 19 (12), 1572-1574 (2003).</li><li>Altschul, S. 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=Gapped+BLAST+and+PSI-BLAST:+a+new+generation+of+protein+database+search+programs.">Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.</a> Nucleic Acids Res. 25 (17), 3389-3402 (1997).</li><li>Zhang, Z., 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=Protein+sequence+similarity+searches+using+patterns+as+seeds.">Protein sequence similarity searches using patterns as seeds.</a> Nucleic Acids Res. 26 (17), 3986-3990 (1998).</li></ol>

命名组内的蛋白质标准应该是明确的但事实并非总是如此。在许多实验室使用各种各样的生物，导致在不同系统中的术语，以及不同程度的表征，研究了有 CS-α β 折叠的序列。企图强加一个完全新的命名法是不合理的会导致大量的混乱时以前文献。参考编号系统可以蛋白质名称用于澄清其与家族的特征。
组蛋白的明确标准的命名和分类将不可能受益于生成参考在电子表格中?...

蛋白质的名称应该反映是特征及与其他蛋白质的关系。不幸的是，名称通常会发现当时被分配，并且，随着研究的继续，较大范围的理解可能更改。如果蛋白质被独立地识别由多个实验室，变化在命名或被认为是明确分配名称时的特点和充足地不再区分蛋白质的名称，这会导致多个名称从别人。
无脊椎动物防御提供很好的例子，变性的命名和分类。第一次的无脊椎动物防御宗从昆虫，和名称"昆虫防御素"，提出了基于感知的同源性为哺乳动物防御素1，2。长期防御素仍然使用，即使它是现在很清楚那无脊椎动物及哺乳动物防御素不共享一个共同的祖先3，4。根据物种，"防御"无脊椎动物可能有六个或八个半胱氨酸 （即形成三个或四个二硫键） 和各种抗菌活性。防止局势进一步复杂化，具有相同特征的蛋白质称为防御素是不总是"防御，"如最近发现的 cremycins 从秀丽 remanei5。此外，无脊椎动物大防御素是更有可能进化有关脊椎动物 β-防御素比到其他无脊椎动物防御6。尽管这样，研究人员有时依赖名称"防御"确定哪些序列应列入分析时。
结构的研究揭示昆虫防御素与蝎子毒素7，之间的相似性和 CS-α β 折叠随后成立作为昆虫防御8的定义结构特征。这折叠结构蛋白质分类 (SCOP) 数据库中9，目前包括五个家庭定义蝎子毒素样 （CS-α β） 超家族： 昆虫防御素、 短链蝎毒素、 长链蝎毒素，MGD 1 （从软体动物） 和植物防御素。这个家族是最近描述的独联体防御4和蛋白酶基因 3D 数据库10，11超 3.30.30.10 家族的代名词。从繁多的无脊椎动物类群、 植物和真菌显示的名称包含这折叠的蛋白质显然无关的半胱氨酸数量的抗菌活性或键合模式、 进化历史12的研究。
缺乏一致性和明确的标准，使它具有挑战性进行命名和分类在这个家族中的新发现序列。比较此超家族蛋白的主要障碍是半胱氨酸的编号对每个单独序列 （每个序列中的第一个半胱氨酸是 C1），没有办法解释的结构的作用。这意味着只有序列与相同数量的半胱氨酸可以进行比较。还有小序列保守性非半胱氨酸形成 CS-α β 折叠，使线路及系统发育分析困难。通过开发划分结构功能优先级编号系统，可以更轻松地比较和对齐家族序列。保守的特点，以及那些定义分组，可以快速，可视化和新序列可以更轻松地放入适当的上下文。
本文使用一个电子表格软件 （如Excel） 来生成参考编号系统的 CS-α β 超家族。它显示如何这澄清序列之间的比较，并将其应用于新的 CS-α β 序列确定从缓步动物。使用 CS-α β 超家族作为一个例子，是写议定书 》 提供指导，当使用序列的兴趣;然而，它不是要具体到这个家族或富含半胱氨酸序列。此方法可能会最有用的蛋白质，有独立研究在不同类群和 （或） 有小的整体序列同源性，与离散性的特征，不可能很容易识别的分子分析软件组。此方法要求一些先验的决定，有关重要的功能，所以它将有限的公共事业，如果没有重要的特点，确定了。主要的目标是展示如何可以实现一个简单的可视化的序列关系。这然后可以用于通知序列比对和分析，但如果对齐方式和分析的主要目标，条形码方法会有更多的容量，为自动化13个合适的选择。当前方法的线性形式，显示每个肽的特点，所以它不会有助于直接可视化的三维结构。

<table width="573">
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	<tbody>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">BLAST webpage</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">https://blast.ncbi.nlm.nih.gov/Blast.cgi</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">EditSeq (Lasergene suite)</td>
			<td style="width:71px;">DNASTAR</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">https://www.dnastar.com/t-allproducts.aspx</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Excel 2013</td>
			<td style="width:71px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:216px;height:63px;">FigTree</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">&#160;http://tree.bio.ed.ac.uk/software/figtree/</td>
		</tr>
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			<td height="21" style="width:216px;height:21px;">MEGA</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>www.megasoftware.net</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">MrBayes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">http://mrbayes.sourceforge.net/</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">SCOP database</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">http://scop.mrc-lmb.cam.ac.uk/scop/</td>
		</tr>
	</tbody>
</table>

creating and applying a reference to facilitate the discussion and classification of proteins in a diverse group

研究了在不同的实验室使用不同生物体的相关的蛋白可能缺乏统一的制度的命名和分类，制作困难，讨论组作为一个整体并放置到适当的上下文的新序列。发展优先重要序列特征的引用有关的结构和 （或) 活动可用于除规定名称向不同的蛋白质组中添加一些相干。本文使用的半胱氨酸稳定 α 螺旋 （CS-α β） 超家族作为一个例子显示如何在电子表格软件中生成的引用可以澄清现有蛋白超家族成员之间的关系，以及促进新的加法序列。它还演示如何引用可以帮助完善中常用的软件，系统发育分析的有效性的影响生成的序列比对。引用的使用可能会对于蛋白质组包含高度不同的序列，从广泛的类群，具有各种功能，不充分的分子分析捕捉到最有帮助。

序列在文献报道的 CS-α β 超家族中的组如图 4所示。半胱氨酸配对的基础，每个序列的编号表明五个基本组 (表 1，中间列)。第一小组有六个半胱氨酸，从三个二硫键债券，包括昆虫、 蛛形纲动物、 软体动物、 线虫、 真菌从序列。组 2、 3 和 4 已形成四个二硫键的 8 半胱氨酸。第二组包括昆虫、 蜘蛛纲动物和植物序列;第 3 组包括蛛形?...

Watch this Scientific Journal Video about Creating and Applying a Reference to Facilitate the Discussion and Classification of Proteins in a Diverse Group at JoVE.com

Creating and Applying a Reference to Facilitate the Discussion and Classification of Proteins in a Diverse Group

创建和应用的参考，以便不同的组中的蛋白质分类与探讨

本议定书的目标是制定一个基准不同蛋白质缺乏连贯一致的命名和分类标准的组中。此引用有助于分析和小组讨论作为一个整体，可采用除规定名称。

Related proteins that have been studied in different labs using varying organisms may lack a uniform system of nomenclature and classification, making it ...

The overall goal of this procedure is to use standard spreadsheet software to develop a reference for divergent proteins in a group that lacks coherent criteria for nomenclature and classification. This method can help clarify relationships between related proteins that have confusing or inconsistent nomenclature, such as the cysteine-stabilized alpha-beta superfamily of defensive peptides that includes invertebrate defenses. The main advantage of this technique is that it provides a simple visual representation of the proteins of interest.To begin, identify the defining characteristics of the protein group of interest. For example, the CS alpha-beta fold in the solution structure of insect defense and aid from phormia terraenovae defines the CS alpha-beta superfamily. This fold also includes a smaller motif called the cysteine-stabilized helix, which is identified by CXXXC upstream of a CXC.The four cysteines form two disulfide bonds. To complete the CS alpha-beta motif, a third disulfide bond is formed by an additional pair of cysteines. Ultimately it is critical to know at least some important features related to the structure or function of the protein.Without this, there is no basis for generating a reference. Now, enter these defining features into a spreadsheet. Use columns for the conserved features and to represent the spaces between these features.Keep the columns wide enough to fit numbers, and give them a consistent width. In the rows, describe the sequences. To indicate a sequence as a feature, fill in the feature box with color, using the fill function.Then, to indicate the spacing between the features, enter the number of amino acids in the box between. Now, add representative sequences that have been previously established as members of the group based on structural databases and published results. As needed, add features that are likely to define a subgroup of sequences, such as an extra cysteine.If features are missing from a given sequence, leave the box unfilled and combine it with boxes representing intervening amino acids. To do this, us the Merge and Center function. Once representative sequences have been entered, identify groups of clearly related sequences.Then, summarize the characteristics of these groups. When the number of amino acids between features varies, use a hyphen to indicate a range or slashes to indicate a few specific numbers. As needed, creatively annotate features that may be relevant or aren't common enough to include in the reference.For example, since cysteines are important in the superfamily, the presence of additional cysteines can be labeled. Sometimes, when adding newly identified sequences to the spreadsheet, the sequences of one species fall into several different groups of the superfamily, such as for tardigrades. Once completed, the rows of the spreadsheet can then be sorted to highlight variation within a species, or variation in between taxonomic groups.This demonstration uses the freely available MEGA 6 software. However, other software can be used similarly. To start a new alignment, select Edit/Build Alignment under the Align tab.Then select Create a new alignment, in the box that appears and click OK.Then select Protein. Now, select Insert Sequence from File, in the Edit menu, to import the sequences. The sequences must be in FASTA format.Background colors reflecting different amino acid types are shown by default and can be turned off with a toggle, under the Display menu. Once all the sequences are entered, click the flexing arm icon and then Align Protein to align the sequences using the muscle algorithm. If the message, Nothing selected for alignment.Select all? pops up, choose OK.Some parameters can be changed in the pop-up window, but for this demonstration, the defaults will suffice. Now, check the alignment based on the important features of the protein superfamily.The top bar shows an asterisk in any position in which the amino acid is completely conserved. The initial alignment identifies three of the four conserved cysteines. One sequence is clearly misaligned.To fix a misaligned sequence, highlight the dashes and press the Delete key without accidentally deleting amino acids. Then, move the amino acids into their proper alignment by adding spaces. After aligning the sequence manually, note that the last cysteine of the CXXXC motif is conserved throughout the alignment.Manual adjustment is often necessary to prioritize the most important features of the sequences. The spreadsheet alignment of the previously established CS alpha-beta superfamily revealed five basic patterns of bond formation. The newly identified tardigrade sequences fell into the complete spectrum of these patterns.Next, phylogenetic analyses were used to examine how this group of proteins may have evolved. However, the sequences are generally short and highly divergent. Thus, the resulting trees were poorly resolved and offered little insight.The multiple sequence alignment was optimized using the reference, but there was still poor resolution in the maximum likelihood analysis and in the Bayesian phylogenetic analysis. Most clades had only low levels of support. However, five small groups were supported in at least one of the two trees.Sequences with different cysteine numbers within a taxonomical group may be more closely related than sequences with the same pattern from different groups. After watching this video, you should have a good understanding of how to use spreadsheet software to generate a simple visualization of important protein characteristics. While attempting this procedure, it's important to remember that determining the most relevant characteristics for the group of proteins is often an iterative process, and revision of the reference will likely be necessary.New sequences can always be added to a spreadsheet, and it is easy to have multiple versions allowing for new features to be added that may be useful for classification and analysis. Although the spreadsheet alignment allows easy visualization of structural and functional features, other methods like phylogenetic analysis can be performed to provide additional insight into evolutionary relationships.

Research

JoVE Journal

Behavior

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

一致性的任务作为一种手段来获取目标行为的措施中的橡胶手错觉范式Crossmodal

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

科研

行为学

Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat

使用威胁概率任务，以评估焦虑和恐惧在不确定性和一定的威胁

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

修改后的孔板 - 测量行为，认知和社会互动小鼠和大鼠

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

应用立体定向注射技术在研究动物行为的遗传效应

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

应力的社会问题：社会支持和社会认同的特里尔社会的实验操作压力测试

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

Psychophysical Tracking Method to Measure Taste Preferences in Children and Adults

心理跟踪方法来衡量儿童和成人的口味偏好

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to ...

Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

行为障碍：一个创新的方法来监控保健品饮食的调节作用

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, ...

Using a Virtual Store As a Research Tool to Investigate Consumer In-store Behavior

使用虚拟商店作为调查消费者店内行为的研究工具

People&#39;s responses to products and/or choice environments are crucial to understanding in-store consumer behaviors. Currently, there are various ...

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

利用fishsim动画工具链调查鱼类行为--以鱼翅钼中的选物复制为例

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the ...

Applying an eMASS Customization Program as a Research Tool to Evaluate Consumer Benefits

应用 eMASS 定制计划作为评估消费者权益的研究工具

As many scholars and practitioners study personalization and relationship marketing, it is important to provide personalization such as mass customization ...