Name: crystal structure of the n-terminal domain of ryanodine receptor from plutella xylostella
Uploaded: 2018-11-30T14:45:00
Description: Watch this Scientific Journal Video about Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella at JoVE.com

这项研究的资金由以下机构提供: 国家重点研究和发展计划 (2017yfd0201400, 2017YFD0201400)、中国国家自然科学基金 (13320103922, 31230061) 和国家基础研究项目 (973)中国 (2015cb856500, 2015cb856504)。我们感谢上海同步辐射设施 (ssrf) 波束线 bl17u1 的工作人员。

1. 基因克隆、蛋白质表达和纯化
<ol><li>pcr 扩增与感兴趣的蛋白质相对应的 dna (dbm ryr 的残留量 1-205, genbank acc. no。afw97408) 和克隆到 pet-28a-hmt 矢量由连接独立克隆 (lic)25。该载体包含一个组氨酸标记、mbp 标记和 n-终点15处的 tev 蛋白酶裂解位点.
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<li>设计用于扩增目标基因的 lic 引物, 其与 lic 兼容的 5 ' 扩展: 正向 lic 底漆: 5 ' tactcacccatcatgggggggg...

<ol>
	<li>Giannini, G., Sorrentino, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+structure+and+tissue+distribution+of+ryanodine+receptors+calcium+channels.">Molecular structure and tissue distribution of ryanodine receptors calcium channels.</a> Medicinal Research Reviews. 15 (4), 313-323 (1995).</li><li>Takeshima, H., 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=Isolation+and+characterization+of+a+gene+for+a+ryanodine+receptor/calcium+release+channel+in+Drosophila+melanogaster.">Isolation and characterization of a gene for a ryanodine receptor/calcium release channel in Drosophila melanogaster.</a> FEBS Letters. 337 (1), 81-87 (1994).</li><li>Sattelle, D. B., Cordova, D., Cheek, T. 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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=Crystal+structure+of+type+I+ryanodine+receptor+amino-terminal+beta-trefoil+domain+reveals+a+disease-associated+mutation+&amp;#34;hot+spot&amp;#34;+loop.">Crystal structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation &amp;#34;hot spot&amp;#34; loop.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (27), 11040-11044 (2009).</li><li>Lobo, P. 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G., Leitner, A., Aebersold, R., Raunser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+conformational+switch+mechanism+of+the+ryanodine+receptor.">Architecture and conformational switch mechanism of the ryanodine receptor.</a> Nature. 517 (7532), 39-43 (2015).</li><li>Peng, W., 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=Structural+basis+for+the+gating+mechanism+of+the+type+2+ryanodine+receptor+RyR2.">Structural basis for the gating mechanism of the type 2 ryanodine receptor RyR2.</a> Science. 354 (6310), (2016).</li><li>Wei, R. S., 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=Structural+insights+into+Ca2+-activated+long-range+allosteric+channel+gating+of+RyR1.">Structural insights into Ca2+-activated long-range allosteric channel gating of RyR1.</a> Cell Research. 26 (9), 977-994 (2016).</li><li>Yan, 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=Structure+of+the+rabbit+ryanodine+receptor+RyR1+at+near-atomic+resolution.">Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution.</a> Nature. 517 (7532), 50-55 (2015).</li><li>Zalk, R., 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=Structure+of+a+mammalian+ryanodine+receptor.">Structure of a mammalian ryanodine receptor.</a> Nature. 517 (7532), 44-49 (2015).</li><li>Furlong, M. J., Wright, D. J., Dosdall, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diamondback+moth+ecology+and+management:+problems,+progress,+and+prospects.">Diamondback moth ecology and management: problems, progress, and prospects.</a> Annual Review of Entomology. 58, 517-541 (2013).</li><li>Amador, F. 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=Crystal+structure+of+type+I+ryanodine+receptor+amino-terminal+beta-trefoil+domain+reveals+a+disease-associated+mutation+&amp;#34;hot+spot&amp;#34;+loop.">Crystal structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation &amp;#34;hot spot&amp;#34; loop.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (27), 11040-11044 (2009).</li><li>Lobo, P. 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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=Phaser+crystallographic+software.">Phaser crystallographic software.</a> Journal of Applied Crystallography. 40 (Pt 4), 658-674 (2007).</li><li>Adams, P. 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=PHENIX:+a+comprehensive+Python-based+system+for+macromolecular+structure+solution.">PHENIX: a comprehensive Python-based system for macromolecular structure solution.</a> Acta Crystallographica Section D. 66 (Pt 2), 213-221 (2010).</li><li>Zwart, P. H., Gross-Kunstleve, R. W., Adams, P. 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V., 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=Towards+automated+crystallographic+structure+refinement+with+phenix.refine.">Towards automated crystallographic structure refinement with phenix.refine.</a> Acta Crystallographica Section D. 68 (Pt 4), 352-367 (2012).</li><li>Lin, L., 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=Crystal+structure+of+ryanodine+receptor+N-terminal+domain+from+Plutella+xylostella+reveals+two+potential+species-specific+insecticide-targeting+sites.">Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites.</a> Insect Biochemistry and Molecular Biology. 92, 73-83 (2018).</li><li>Qi, S., Casida, J. 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本文描述了 dbm ryr ntd 的重组表达、纯化、结晶和结构的确定过程。对于结晶, 一个关键的要求是获得高溶解度、纯度和均匀性的蛋白质。在我们的协议中, 我们选择使用 pet-28a-hmt 矢量, 因为它包含一个六氨酸标记和 mbp 标记, 这两个标记都可用于纯化, 以获得更高的折叠纯度。此外, mbp 标记有助于目标蛋白的溶解度。我们通过连续五个步骤纯化蛋白质, 得到了高度纯净、适合结晶的蛋白质。采用自动...

ryanodine 受体 (ryr) 是特定的离子通道, 它介导ca 2 +离子在肌肉细胞中的肉瘤-质网膜 (sr) 的渗透。因此, 它们在激励收缩耦合过程中发挥着重要作用。在其功能形式中, ryr 组装成一个同型四聚体, 其分子质量为 gt;2 mda, 每个亚基由 ~ 5000个氨基酸残基组成。在哺乳动物中, 有三种等形式: ryr1-骨骼肌类型, ryr2-心肌类型和 ryr3-在不同的组织1中普遍表达。
在昆虫中只有一种类型的 ryr, 它表现在肌肉和神经组织2中。昆虫 ryr 更类似于哺乳动物 ryr2, 序列特征约为 47%3。拜耳 (氟苯二胺)、杜邦 (氯硝酸) 和先正达 (氰基吡鱼) 等主要公司开发并销售了针对舌再产的二铵杀虫剂。自最近推出以来, 二胺类杀虫剂已成为增长最快的杀虫剂类别之一。目前, 这三种杀虫剂的年销售额已超过20亿美元, 增长率自2009年以来超过 50% (阿特拉诺娃)。
最近的研究报告了在使用这些杀虫剂4、5、6、7、8几代之后昆虫抗药性的发展。来自钻石蛾 (dbm)、 plutella xylostella ( g4946e, i4790m) 的 ryr 跨膜域的抗性突变以及番茄叶矿工tuta aicuruta的相应位置表明该地区可能与二胺杀虫剂结合, 因为该地区是众所周知的关键是门控通道 4,8,9。尽管在这一领域进行了广泛的研究, 但二胺类杀虫剂的确切分子机制仍然难以捉摸。此外, 目前尚不清楚耐药突变是直接影响与二胺的相互作用还是同种异体的相互作用。
早期的研究报告了哺乳动物种的几个 ryr 域的结构, 以及 x 射线晶体学和低温电子显微镜分别为 10、11、 12、13、14、15、16、17、18、19、20、21.但到目前为止, 还没有报道昆虫 ryr 的结构, 这使得我们无法了解受体功能的分子复杂性, 以及杀虫剂作用和杀虫剂抗药性的发展的分子机制。
在这篇手稿中, 我们提出了一个广义的协议的结构表征 n 端β-树箔域的 ryanodine 受体从钻石蛾, 一种破坏性的害虫感染世界各地的十字花科作物22。该结构是根据出版的兔子 ryr1 ntd 晶体结构23,24和低温 em 结构模型16,17,18, 19设计的。20,21. 这是为昆虫 ryr 报告的第一个高分辨率结构, 它揭示了通道门控的机制, 并为利用结构药物设计开发物种专用杀虫剂提供了一个重要模板。在结构阐明方面, 我们采用了 x 射线晶体学, 它被认为是在近原子分辨率下测定蛋白质结构的 "金标准"。虽然结晶过程是不可预测的和劳动密集型的, 这一步一步的协议将帮助研究人员表达, 纯化和表征昆虫 ryr 或任何其他蛋白质的其他领域或一般。

<table border="0" cellpadding="0" cellspacing="0" width="1450">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pET-28a-HMT vector</td>
			<td style="width:164px;"></td>
			<td style="width:129px;"></td>
			<td style="width:941px;">This modified pET vector contains a hexahistidine tag, an MBP fusion protein and a TEV protease cleavage site at the N-terminus (Lobo and Van Petegem, 2009)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E. coli BL21 (DE3) strain</td>
			<td style="width:164px;">Novagen</td>
			<td>69450-3CN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HisTrapHP column (5 mL)</td>
			<td>GE Healthcare</td>
			<td>45-000-325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amylose resin column</td>
			<td>New England Biolabs</td>
			<td style="width:129px;">E8021S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q Sepharose high-performance column&#160;</td>
			<td>GE Healthcare</td>
			<td>17-1154-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon concentrators (10 kDa MWCO)</td>
			<td>Millipore</td>
			<td>UFC901008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superdex 200 26/600 gel-filtration column&#160;</td>
			<td>GE Healthcare</td>
			<td>28-9893-36</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Automated liquid handling robotic system&#160;</td>
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		<tr height="21">
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			<td>82050-494</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Uni-Puck</td>
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			<td height="21" style="height:21px;">Mounted CryoLoop - 20 micron</td>
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			<td height="21" style="height:21px;">CryoWand</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Puck dewar loading tool</td>
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			<td height="21" style="height:21px;width:216px;">Nano drop</td>
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			<td height="21" style="height:21px;width:216px;">Plasmid mini-prep kit</td>
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			<td height="21" style="height:21px;width:216px;">Gel extraction kit</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:164px;">Genview</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol</td>
			<td style="width:164px;">Genview</td>
			<td align="right" style="width:129px;">60242</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
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			<td height="21" style="height:21px;width:216px;">Sonnicator</td>
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			<td height="21" style="height:21px;width:216px;">Protein purification system</td>
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			<td height="21" style="height:21px;width:216px;">Light microscope</td>
			<td style="width:164px;">Nikon</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IzIt crystal dye</td>
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			<td>HR4-710</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrophoresis unit</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:129px;">1658005EDU</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaker Incubator</td>
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			<td height="21" style="height:21px;width:216px;">Index crystal screen</td>
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			<td style="width:129px;">HR2-144</td>
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		</tr>
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			<td height="21" style="height:21px;width:216px;">Structure crystal screen</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ProPlex crystal screen</td>
			<td style="width:164px;">Molecular Dimensions</td>
			<td style="width:129px;">MD1-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PACT premier crystal screen</td>
			<td style="width:164px;">Molecular Dimensions</td>
			<td style="width:129px;">MD1-29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">JCSG-plus crystal screen</td>
			<td style="width:164px;">Molecular Dimensions</td>
			<td style="width:129px;">MD1-37</td>
			<td></td>
		</tr>
	</tbody>
</table>

crystal structure of the n-terminal domain of ryanodine receptor from plutella xylostella

开发针对昆虫赖诺丁受体 (ryr) 的有效和高效杀虫剂在农业虫害防治领域引起了极大的兴趣。迄今为止, 几种针对害虫 ryr 的二胺类杀虫剂已经商业化, 年收入达20亿美元。但由于缺乏有关昆虫 ryr 的结构信息, 对 ryr 靶向杀虫剂作用方式的理解受到限制。这反过来又限制了对害虫中杀虫剂抗药性发展的理解。钻石返回蛾 (dbm) 是一种破坏世界各地十字花科作物的破坏性害虫, 据报道, 这种害虫对菱形杀虫剂也表现出抗药性。因此, 开发针对 dbm ryr 的新型杀虫剂, 特别是针对不同于传统二胺结合位点的地区, 具有十分重要的现实意义。在这里, 我们提出了一个协议, 从结构上描述 n 终端域的 ryr 从 dbm。x 射线晶体结构是通过分子置换求解的 2.84, 该分辨率呈β-树花药折叠图案和侧翼α螺旋。该协议可用于其他领域或一般蛋白质的表达、纯化和结构表征。

净化
dbm ryr 的 n 端域被表示为具有六角蛋白标记、mbp 标记和 tev 蛋白酶裂解位点的融合蛋白。我们遵循五步纯化策略, 以获得一种高度纯的蛋白质, 适合结晶目的。首先, 用 ni-nta 柱 (histrap hp) 从细胞裂解液的可溶性组分中纯化了融合蛋白。其次, 对融合蛋白进行 tev 蛋白酶裂解, 用直链淀粉树脂柱去除六角蛋白 mbp-多重症, 然后采用?...

Watch this Scientific Journal Video about Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella at JoVE.com

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

本文介绍了小菜蛾赖诺酮受体 n 端域的蛋白表达、纯化、结晶和结构测定的方案.

龙苷受体 n 端域的晶体结构

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

解决瑞安碘受体域的结构有助于我们了解蛋白质功能的分子机制、杀虫剂作用和杀虫剂耐药性的发展。这一措施意味着使用X射线晶体学进行结构图示，这被认为是以近原子分辨率测定蛋白质结构的黄金标准。这种昆虫Ryanodine受体域的高分辨率结构揭示了通道门控的机理，为利用基于结构的药物设计方法开发物种特异性杀虫剂提供了重要的模板。一般来说，对这种方法的新鲜人会很吃苦，因为未来的蛋白质晶体学家必须精通生物化学、生物物理学、计算机科学和数学。首先通过聚合酶链反应放大与感兴趣的蛋白质对应的DNA。在反应结束时，在2%的阿加罗斯凝胶上运行整个50微升的反应混合物，然后根据标准协议用凝胶提取试剂盒提取DNA。接下来，在30微升双蒸馏水中，将20微升的载体DNA与6微升10倍反应缓冲液和4微升SSPI限制酶混合，使LIC载体DNA线性化。在37摄氏度下孵育这种反应混合物3小时。在孵化结束时，在1%的阿加罗斯凝胶上运行整个反应混合物，然后根据制造商的说明使用凝胶提取试剂盒提取线性化载体DNA。接下来，根据标准协议，对线性化载体DNA和PCR放大插入DNA的5微升进行独立的T4 DNA聚合酶处理。在室温下孵育40分钟，随后在75摄氏度下孵育20分钟的酶热灭活。通过将 T4 处理的 2 微升插入 DNA 与 T4 处理 LIC 载体 DNA 的 2 微升结合，在室温下孵育 10 分钟，执行与结扎无关的克隆退火反应。将BL21（DE3）大肠杆菌与重组质粒进行转化，在冰上解冻50微升的称职细胞，并将约1微升的退火质粒加入管中。轻轻轻拂管子两到三次，将细胞和DNA混合，将管子放回冰上20分钟。在孵化结束时，在42摄氏度的温度下热击细胞40秒，然后回到冰上两分钟。接下来，在管中加入一毫升室温 LB 介质，在 37 摄氏度下将细胞放在 250 RPM 摇床上 45 分钟。在震动孵育结束时，将这种混合物的150至200微升板放入选择板，并在37摄氏度下将板在37摄氏度下过夜。第二天，在37摄氏度的摇床孵化器中，在100毫升的2-YT介质中培养一个群体，辅以卡那霉素。第二天早上，接种一升2-YT介质，辅以卡那霉素，10毫升过夜培养，并在37摄氏度下孵育，摇晃，直到OD600读数达到约0.6。然后，诱导与IPTG培养物最终浓度为0.4毫摩尔，并在30摄氏度下生长细胞5小时。在孵化结束时，通过离心收获细胞，并每40毫升的解液缓冲液浓度将颗粒重新悬浮到10克细菌。以 65% 振幅的声波破坏细胞壁，在一秒关闭一秒时中断细胞壁，8 分钟。通过离心去除细胞碎屑。然后，通过 22 微米过滤器过滤上清液，然后将溶液加载到样品循环中。为了净化融合蛋白，将样品环中过滤的上清液注射到纯化系统中的5毫升镍-硝基色色柱中，其线性梯度为20至250毫摩尔。用烟草蚀刻病毒蛋白酶分离的分离目标蛋白，以1-50的比例，在4摄氏度过夜，并纯化了阿米洛塞树脂柱上的裂解反应混合物和镍硝基蛋白柱，去除标签和烟草蚀刻病毒蛋白酶。将透析缓冲液从镍-硝基色柱中透析的流经中解入，以降低盐浓度，并在洗脱缓冲液中用20至500毫摩尔氯化钾的线性梯度净化苯乙烯交换柱上的样品。然后，将蛋白质浓缩在离心浓缩器中。将浓缩蛋白注入 Superdex 200 26/600 凝胶过滤柱，以检查均匀性，并评估蛋白质的纯度 15%SDS-PAGE。为了准备结晶的蛋白质，将纯化蛋白质样品浓缩到离心浓缩器中每毫升10毫克，并在80摄氏度储存前将缓冲液交换到结晶缓冲液中。要进行结晶筛选，请使用 295 Kelvin 的坐滴蒸汽扩散方法，并采用多个结晶套件，以及 96 井格式的自动液体处理系统。在落液设置结束时，密封 96 井结晶板以防止蒸发，并实现储液罐缓冲液内蛋白质滴的均衡。将板放在 18 摄氏度的晶体培养箱中。在光学显微镜下定期检查板，以监测晶体的形成和生长。为了将蛋白质晶体与盐晶体区分开来，在目标滴中加入一微升蛋白质晶体染料，并在一小时后在显微镜下观察晶体。蛋白质晶体会出现蓝色。为了进一步优化晶体，在24孔板中采用正结晶条件和悬挂滴蒸汽扩散法，随后在18摄氏度的晶体孵化器中孵育。要确定蛋白质结构，请将晶体安装在光显微镜下，用于液氮中的闪光冷却，然后将晶体放在单桶中进行储存和运输。在内部 X 射线衍射器中预筛选晶体，使用手动居中功能安装和居中晶体。然后，使用 X 射线衍射软件收集数据。要索引、集成和缩放数据集，使用 HKL-2000 套件，首先选择探测器并加载数据集。然后，执行峰值搜索功能，查找衍射点。索引点，选择正确的空间组，并执行峰值集成。接下来，缩放数据集。调整错误模型并再次缩放。保存输出 sca 文件。若要使用 Phenix 软件套件确定结构，请创建一个新项目。首先，使用 sca 文件运行 Extriage，以计算非对称单元中蛋白质分子的可能拷贝数。其次，通过分子置换、运行相位器解决相位问题，利用衍射数据文件、具有高序列标识和结构相似性作为目标蛋白的模板结构文件以及蛋白质序列文件，找到解决方案。使用 Phaser 的输出文件和目标蛋白质的序列文件，在 Phenix 中执行自动构建以生成初始模型。使用COOT手动将结构构建成经过修改的实验电子密度，并使用 Phenix 在迭代周期中细化。使用 Phenix 中的验证工具验证最终模型。在这个具有代表性的实验中，通过STS-PAGE分析，对金刚背飞蛾ryanodine受体蛋白的终端域的纯化，通过STS-PAGE分析，产生了一个带，大约21千吨。凝胶过滤柱的洗脱体积确认纯化的ryanodine受体终端域为单体。对于结晶，形成高质量板状晶体的最佳条件是在存在1个pH值为7的摩尔 HEPES 和1.6个硫酸铵。通过演示的衍射数据集对蛋白质结构的测定揭示了覆盖残留物1至205的金刚背飞蛾ryanodine受体终端域。具有大紊乱、灵活区域或弱亲缘的蛋白质很难结晶。在这些情况下，蛋白质工程策略，如表面熵减少，循环截断和交叉链接，可能会增加获得更好的蛋白质晶体的可能性。除了揭示高分辨率蛋白质结构外，X射线晶体学还可用于研究蛋白质-农药相互作用，这可能有助于基于结构的农药设计。

Research

JoVE Journal

Biochemistry

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

Immunization of Alpacas Lama pacos with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

蛋白质抗原对羊的免疫和抗原特异性单域抗体的制备

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies ...

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

利用网格和收集荧光蛋白陶瓷的结构解

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...