Name: expression and purification of the human lipid-sensitive cation channel trpc3 for structural determination by single-particle cryo-electron microscopy
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Description: Watch this Scientific Journal Video about Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy at JoVE

我们感谢赵先生和孟先生在大卫·范安德尔高级冷冻电子显微镜套房的数据收集方面给予的支持。我们感谢 vari 高性能计算团队提供的计算支持。我们感谢 n. clemente、d. dues、j. floramo、y. 黄、y. kim、c. mueller、b. roth 和 z. ruan 提出的意见, 这些评论大大改进了这份手稿。我们感谢 d. nadziejka 对这份手稿的编辑支持。这项工作得到了 vari 内部资金的支持。

1. dh10α组成细胞转化为 bacm中 dna
<ol><li>合成感兴趣的基因, 并将其亚化为一个修改版本的 peg 向量, 其中包含一个双链球标记、一个 his8 标记和 gfp, 在 n 终点 (pfastbaci) 20 处有凝血酶裂解部位。</li>
	<li>通过在 1.5 ml 管中加入5纳克含有所需基因的质粒, 在 pFastBacI 中加入50μl 的 dh10α细胞, 并在冰上孵育 10分钟, 从而转化合格的细胞。在42°c 下, 热冲击细胞45秒。在管中加入200μl 的超最...

<ol>
	<li>Berridge, M. J., Bootman, M. D., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling:+dynamics,+homeostasis+and+remodelling.">Calcium signalling: dynamics, homeostasis and remodelling.</a> Nature Reviews Molecular Cell Biology. 4 (7), 517-529 (2003).</li><li>Kumar, R., Thompson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+parathyroid+hormone+secretion+and+synthesis.">The regulation of parathyroid hormone secretion and synthesis.</a> Journal of the American Society of Nephrology. 22 (2), 216-224 (2011).</li><li>Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+control+of+neurotransmitter+release.">Calcium control of neurotransmitter release.</a> Cold Spring Harbor Perspectives in Biology. 4 (1), a011353 (2012).</li><li>Ong, H. L., de Souza, L. B., Ambudkar, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+TRPC+Channels+in+Store-Operated+Calcium+Entry.">Role of TRPC Channels in Store-Operated Calcium Entry.</a> Advances in Experimental Medicine and Biology. 898, 87-109 (2016).</li><li>Smyth, J. T., 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=Activation+and+regulation+of+store-operated+calcium+entry.">Activation and regulation of store-operated calcium entry.</a> Journal of Cellular and Molecular Medicine. 14 (10), 2337-2349 (2010).</li><li>Prakriya, M., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-Operated+Calcium+Channels.">Store-Operated Calcium Channels.</a> Physiological Reviews. 95 (4), 1383-1436 (2015).</li><li>Liu, X., Singh, B. B., Ambudkar, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC1+is+required+for+functional+store-operated+Ca2++channels.+Role+of+acidic+amino+acid+residues+in+the+S5-S6+region.">TRPC1 is required for functional store-operated Ca2+ channels. Role of acidic amino acid residues in the S5-S6 region.</a> Journal of Biological Chemistry. 278 (13), 11337-11343 (2003).</li><li>Zhu, X., Jiang, M., Birnbaumer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-activated+Ca2++influx+via+human+Trp3+stably+expressed+in+human+embryonic+kidney+(HEK)293+cells.+Evidence+for+a+non-capacitative+Ca2++entry.">Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry.</a> Journal of Biological Chemistry. 273 (1), 133-142 (1998).</li><li>Zhu, X., 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=trp,+a+novel+mammalian+gene+family+essential+for+agonist-activated+capacitative+Ca2++entry.">trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry.</a> Cell. 85 (5), 661-671 (1996).</li><li>Itsuki, K., 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=Voltage-sensing+phosphatase+reveals+temporal+regulation+of+TRPC3/C6/C7+channels+by+membrane+phosphoinositides.">Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides.</a> Channels (Austin). 6 (3), 206-209 (2012).</li><li>Tang, 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=Identification+of+common+binding+sites+for+calmodulin+and+inositol+1,4,5-trisphosphate+receptors+on+the+carboxyl+termini+of+trp+channels.">Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels.</a> Journal of Biological Chemistry. 276 (24), 21303-21310 (2001).</li><li>Gonzalez-Cobos, J. C., Trebak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC+channels+in+smooth+muscle+cells.">TRPC channels in smooth muscle cells.</a> Frontiers in Bioscience (Landmark Edition). 15, 1023-1039 (2010).</li><li>Li, H. S., Xu, X. Z., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+a+TRPC3-dependent+cation+current+through+the+neurotrophin+BDNF).">Activation of a TRPC3-dependent cation current through the neurotrophin BDNF).</a> Neuron. 24 (1), 261-273 (1999).</li><li>Becker, E. 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=Candidate+screening+of+the+TRPC3+gene+in+cerebellar+ataxia.">Candidate screening of the TRPC3 gene in cerebellar ataxia.</a> Cerebellum. 10 (2), 296-299 (2011).</li><li>Kitajima, N., 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=TRPC3+positively+regulates+reactive+oxygen+species+driving+maladaptive+cardiac+remodeling.">TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling.</a> Scientific Reports. 6, 37001 (2016).</li><li>Yang, S. L., Cao, Q., Zhou, K. C., Feng, Y. J., Wang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+receptor+potential+channel+C3+contributes+to+the+progression+of+human+ovarian+cancer.">Transient receptor potential channel C3 contributes to the progression of human ovarian cancer.</a> Oncogene. 28 (10), 1320-1328 (2009).</li><li>Oda, K., 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=Transient+receptor+potential+cation+3+channel+regulates+melanoma+proliferation+and+migration.">Transient receptor potential cation 3 channel regulates melanoma proliferation and migration.</a> Journal of Physiological Sciences. 67 (4), 497-505 (2017).</li><li>Xia, M., Liu, D., Yao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC3:+A+New+Target+for+Therapeutic+Strategies+in+Chronic+Pain-DAG-mediated+Activation+of+Non-selective+Cation+Currents+and+Chronic+Pain+(Mol+Pain+2014;10:43).">TRPC3: A New Target for Therapeutic Strategies in Chronic Pain-DAG-mediated Activation of Non-selective Cation Currents and Chronic Pain (Mol Pain 2014;10:43).</a> Journal of Neurogastroenterology and Motility. 21 (3), 445-447 (2015).</li><li>Fan, C., Choi, W., Sun, W., Du, J., Lu, W. <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+human+lipid-gated+cation+channel+TRPC3.">Structure of the human lipid-gated cation channel TRPC3.</a> Elife. 7, e36852 (2018).</li><li>Goehring, 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=Screening+and+large-scale+expression+of+membrane+proteins+in+mammalian+cells+for+structural+studies.">Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.</a> Nature Protocols. 9 (11), 2574-2585 (2014).</li><li>Hattori, M., Hibbs, R. E., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-detection+size-exclusion+chromatography-based+thermostability+assay+to+identify+membrane+protein+expression+and+crystallization+conditions.">A fluorescence-detection size-exclusion chromatography-based thermostability assay to identify membrane protein expression and crystallization conditions.</a> Structure (London, England: 1993). 20 (8), 1293-1299 (2012).</li><li>Zheng, S. 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=MotionCor2:+anisotropic+correction+of+beam-induced+motion+for+improved+cryo-electron+microscopy.">MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy.</a> Nature Methods. 14 (4), 331-332 (2017).</li><li>Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gctf:+Real-time+CTF+determination+and+correction.">Gctf: Real-time CTF determination and correction.</a> Journal of Structural Biology. 193 (1), 1-12 (2016).</li><li>Scheres, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RELION:+implementation+of+a+Bayesian+approach+to+cryo-EM+structure+determination.">RELION: implementation of a Bayesian approach to cryo-EM structure determination.</a> Journal of Structural Biology. 180 (3), 519-530 (2012).</li><li>Punjani, A., Rubinstein, J. L., Fleet, D. J., Brubaker, 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=cryoSPARC:+algorithms+for+rapid+unsupervised+cryo-EM+structure+determination.">cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination.</a> Nature Methods. 14 (3), 290-296 (2017).</li><li>Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frealign:+An+Exploratory+Tool+for+Single-Particle+Cryo-EM.">Frealign: An Exploratory Tool for Single-Particle Cryo-EM.</a> Methods in Enzymology. 579, 191-226 (2016).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta Crystallographica Section D: Biological Crystallography. 66 (Pt 4), 486-501 (2010).</li><li>Afonine, P. 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: Biological Crystallography. 68 (Pt 4), 352-367 (2012).</li><li>Chen, V. 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=MolProbity:+all-atom+structure+validation+for+macromolecular+crystallography.">MolProbity: all-atom structure validation for macromolecular crystallography.</a> Acta Crystallographica Section D: Biological Crystallography. 66 (Pt 1), 12-21 (2010).</li><li>Scheres, S. H. W., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+overfitting+in+cryo-EM+structure+determination.">Prevention of overfitting in cryo-EM structure determination.</a> Nature Methods. 9 (9), 853-854 (2012).</li><li>Scheres, S. H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RELION:+Implementation+of+a+Bayesian+approach+to+cryo-EM+structure+determination.">RELION: Implementation of a Bayesian approach to cryo-EM structure determination.</a> Journal of Structural Biology. 180 (3), 519-530 (2012).</li><li>Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frealign:+An+Exploratory+Tool+for+Single-Particle+Cryo-EM.">Frealign: An Exploratory Tool for Single-Particle Cryo-EM.</a> Methods in Enzymology. 579, 191-226 (2016).</li><li>Chen, V. 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=MolProbity:+all-atom+structure+validation+for+macromolecular+crystallography.">MolProbity: all-atom structure validation for macromolecular crystallography.</a> Acta Crystallographica Section D-Biological Crystallography. 66, 12-21 (2010).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta Crystallographica Section D-Biological Crystallography. 66, 486-501 (2010).</li><li>Green, E. M., Au, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermostabilization,+Expression,+Purification,+and+Crystallization+of+the+Human+Serotonin+Transporter+Bound+to+S-citalopram.">Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram.</a> Journal of Visualized Experiments. (117), e54792 (2016).</li><li>Mesa, P., Deniaud, A., Montoya, G., Schaffitzel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directly+from+the+source:+endogenous+preparations+of+molecular+machines.">Directly from the source: endogenous preparations of molecular machines.</a> Current Opinion in Structural Biology. 23 (3), 319-325 (2013).</li><li>Bayburt, T. H., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Protein+Assembly+into+Nanodiscs.">Membrane Protein Assembly into Nanodiscs.</a> FEBS letters. 584 (9), 1721-1727 (2010).</li><li>Winkler, P. A., Huang, Y., Sun, W., Du, J., L&#252;, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryo-microscopy+structure+of+a+human+TRPM4+channel.">Electron cryo-microscopy structure of a human TRPM4 channel.</a> Nature. 552, 200-204 (2017).</li><li>Autzen, H. E., 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+human+TRPM4+ion+channel+in+a+lipid+nanodisc.">Structure of the human TRPM4 ion channel in a lipid nanodisc.</a> Science. 359 (6372), 228-232 (2017).</li><li>Guo, 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=Structures+of+the+calcium-activated,+non-selective+cation+channel+TRPM4.">Structures of the calcium-activated, non-selective cation channel TRPM4.</a> Nature. 552 (7684), 205-209 (2017).</li><li>Parmar, 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=Using+a+SMALP+platform+to+determine+a+sub-nm+single+particle+cryo-EM+membrane+protein+structure.">Using a SMALP platform to determine a sub-nm single particle cryo-EM membrane protein structure.</a> Biochimica et Biophysica Acta (BBA)-Biomembranes. 1860 (2), 378-383 (2018).</li><li>Gulati, 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=Detergent-free+purification+of+ABC+(ATP-binding-cassette)+transporters.">Detergent-free purification of ABC (ATP-binding-cassette) transporters.</a> Biochemical Journal. 461 (2), 269-278 (2014).</li></ol>

在过去几年里, 由于开发了新的摄像机和算法, 显著加快了蛋白质的结构测定, 而这些相机和算法并没有显著加快蛋白质的结构测定, 因此, 用低温 em 对蛋白质进行结构测定已经给结构生物学领域带来了革命性的变化。容易结晶, 特别是膜蛋白。尽管冷冻-em 技术最近取得了所有进展, 但在质量和数量上足以促进高质量成像的纯化蛋白的制备通常仍然是耗时、昂贵和具有挑战性的。如上述协议所述, 能?...

钙参与大多数细胞过程, 包括信号级联, 转录控制, 神经递质释放, 和激素分子合成1,2,3。细胞质游离钙的稳态维持对细胞的健康和功能至关重要。细胞内钙稳态的主要机制之一是储存钙进入 (sace), 在这个过程中, 储存在内质网 (er) 中的钙的耗尽会触发质膜上离子通道的打开, 以促进er 钙的补充, 然后可以在进一步信号4,5,6中使用。瞬态受体电位通道 (trpc) 是属于 trp 超家族的透钙通道, 已被确定为 soce7、8、9的主要参与者。
在 trpc 家族的7名成员中, trpc3、trpc6 和 trpc7 构成了同源亚群, 它们在被脂质二级信使二丙烯酸酯 (dag) 激活的能力上是独一无二的, 而二维信使二丙烯酸甘油是脂质信号的降解产物磷脂酰肌醇 4, 5-双磷酸 (pip2)10,11。trpc3 在平滑肌和大脑的大脑和小脑区域中高度表达, 在这些区域中, 它在影响神经传递和神经发生的钙信号中发挥着至关重要的作用。trpc3 功能障碍与中枢神经系统疾病、心血管疾病和某些癌症 (如卵巢腺癌14、15、16) 有关。因此, trpc3 有望成为治疗这些疾病的药物目标。由于对其分子激活机制, 包括脂质结合位点 17、18缺乏了解, 开发针对 trpc3 的有针对性的药物受到限制。我们报告了人类 trpc3 通道 (htrpc3) 及其两个处于封闭状态的脂质结合位点的第一个原子分辨率结构, 为这些机制提供了重要的见解19。
高分辨率膜蛋白结构的关键因素是获得高质量的蛋白质。相应的表达筛选和纯化条件的必要, 以获得高质量的蛋白质可能是一个耗时和昂贵的努力。在这里, 我们提出了一个协议, 详细描述了我们如何确定 htrpc3 的表达和纯化的最佳条件, 它在我们的初始筛选中表现不佳。我们就如何排除故障和优化蛋白质行为提出了几个要点, 为我们的低温电子显微镜 (低温 em) 研究奠定了坚实的基础。我们使用了由 gouaux 和他的同事开发的改良的杆菌病毒产生载体 (peg), 该载体经过优化, 用于在哺乳动物细胞20 中筛选检测和高效生成巴库索病毒。这种表达方法适用于哺乳动物细胞膜中蛋白质的快速和具有成本效益的过度表达。我们将此向量的使用与基于荧光检测大小排除色谱 (fsec) 预置方法21结合起来。该方法采用绿色荧光蛋白 (gfp) 标记融合到感兴趣的结构中, 并提高了目标蛋白在小的全细胞溶解性样品中的可视化。这样就可以在不同的洗涤剂和添加剂的存在下筛选蛋白质稳定性, 并具有稳定的热稳定性突变, 并允许使用少量细胞进行小规模瞬态转染。通过这种方式, 在进入大规模蛋白质纯化之前, 可以快速筛选多种条件。在表达、筛选和纯化之后, 我们提出了一种从低温 em 中获取和处理图像的协议, 以生成蛋白质的新结构测定。我们相信, 这里描述的方法将作为一个通用的协议, 用于 trp 通道受体和其他膜蛋白的结构研究。

<table>
	<tbody>
		<tr>
			<td>pEG BacMam vector (pFastBacI)</td>
			<td>addgene</td>
			<td>31488</td>
			<td></td>
		</tr>
		<tr>
			<td>DH10&#945; cells</td>
			<td>Life Technologies</td>
			<td>10361-012</td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C. media</td>
			<td>Corning</td>
			<td>46003CR</td>
			<td>for transformation of DH10&#945; cells for Bacmid</td>
		</tr>
		<tr>
			<td>Bacmam culture plates</td>
			<td>Teknova</td>
			<td>L5919</td>
			<td>for culture of transformed DH10&#945; cells</td>
		</tr>
		<tr>
			<td>Incubation shaker for bacterial cells</td>
			<td>Infors HT</td>
			<td>Multitron standard</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubated orbital shaker for insect cells</td>
			<td>Thermo-Fisher</td>
			<td>SHKE8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Reach-in CO2 incubator for mammalian cells</td>
			<td>Thermo-Fisher</td>
			<td>3951</td>
			<td></td>
		</tr>
		<tr>
			<td>Table-top orbital shaker</td>
			<td>Thermo-Fisher</td>
			<td>SHKE416HP</td>
			<td>used in Reach-in CO2 incubator for mammalian cells</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>1535</td>
			<td>for bacterial plates</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>for plasmid extraction and purification</td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl alcohol</td>
			<td>Invitrogen</td>
			<td>15593031</td>
			<td>for DNA extraction</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>Life Technologies</td>
			<td>12659017</td>
			<td>insect cells for producing virus</td>
		</tr>
		<tr>
			<td>Sf-900 media</td>
			<td>Gibco</td>
			<td>12658-027</td>
			<td>insect cell media</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellfectin II</td>
			<td>Gibco</td>
			<td>10362100</td>
			<td>for transfecting insect cells</td>
		</tr>
		<tr>
			<td>lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td>for transfecting mamalian cells</td>
		</tr>
		<tr>
			<td>0.2 mm syringe filter</td>
			<td>VWR</td>
			<td>28145-501</td>
			<td>for filtering P1 virus</td>
		</tr>
		<tr>
			<td>0.2 mm filter flasks 500ml resevoir</td>
			<td>Corning</td>
			<td>430758</td>
			<td>for filtering P2 virus</td>
		</tr>
		<tr>
			<td>erlenmeyer culture flask (flat bottom 2L)</td>
			<td>Gene Mate</td>
			<td>F-5909-2000</td>
			<td>for culturing insect cells</td>
		</tr>
		<tr>
			<td>erlenmeyer culture flask (baffled 2L)</td>
			<td>Gene Mate</td>
			<td>F-5909-2000B</td>
			<td>for culturing mammalian cells</td>
		</tr>
		<tr>
			<td>nanodrop 2000 spectrophotometer</td>
			<td>Thermo-Fisher</td>
			<td>ND-2000</td>
			<td>for determining DNA and protein concentrations</td>
		</tr>
		<tr>
			<td>HEK293</td>
			<td>ATCC</td>
			<td>CRL-3022</td>
			<td>mammalian cells for producing protein</td>
		</tr>
		<tr>
			<td>Freestyle 293 expression Medium</td>
			<td>Gibco</td>
			<td>1238-018</td>
			<td>mammalian cell media for protein expression</td>
		</tr>
		<tr>
			<td>Butyric Acid Sodium Salt</td>
			<td>Acros</td>
			<td>263195000</td>
			<td>to amplify protein expression</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Acros</td>
			<td>215740500</td>
			<td>protease inhibitor</td>
		</tr>
		<tr>
			<td>Aprotinin from bovine lung</td>
			<td>Sigma-Aldrich</td>
			<td>A1153-100MG</td>
			<td>protease inhibitor</td>
		</tr>
		<tr>
			<td>Leupeptin hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>24125-16-4</td>
			<td>protease inhibitor</td>
		</tr>
		<tr>
			<td>pepstatin A</td>
			<td>Fisher Scientific</td>
			<td>BP2671-250</td>
			<td>protease inhibitor</td>
		</tr>
		<tr>
			<td>digitonin</td>
			<td>EMD Millipore</td>
			<td>300410</td>
			<td>detergent - to solubilize protein from membrane</td>
		</tr>
		<tr>
			<td>imidazole</td>
			<td>Sigma</td>
			<td>792527</td>
			<td>to elute protein from resin column</td>
		</tr>
		<tr>
			<td>TALON resin</td>
			<td>Clonetech</td>
			<td>635504</td>
			<td>for affinity purification by His-tag</td>
		</tr>
		<tr>
			<td>superose6 incease columns</td>
			<td>GE</td>
			<td>29091596; 29091597</td>
			<td>for HPLC and FPLC</td>
		</tr>
		<tr>
			<td>Prominence Modular HPLC System</td>
			<td>Shimadzu</td>
			<td>See Below</td>
			<td></td>
		</tr>
		<tr>
			<td>Controller Module</td>
			<td>&#34;</td>
			<td>CBM20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Solvent Delivery System</td>
			<td>&#34;</td>
			<td>LC30AD</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Detector</td>
			<td>&#34;</td>
			<td>RF20AXS</td>
			<td></td>
		</tr>
		<tr>
			<td>Autosampler with Cooling</td>
			<td>&#34;</td>
			<td>SIL20ACHT</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure FPLC System with Fractionator</td>
			<td>Akta</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>thrombin (alpha)</td>
			<td>Haematologic Technologies Incorporated</td>
			<td>HCT-0020 Human alpha</td>
			<td>for cleaving GFP tag</td>
		</tr>
		<tr>
			<td>Amicon Ultra 15 mL 100K centrifugal filter tube</td>
			<td>Millipore</td>
			<td>UFC910008</td>
			<td>for concentrating protein</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher</td>
			<td>E478500</td>
			<td>for stabilizing protein</td>
		</tr>
		<tr>
			<td>400 mesh carbon-coated copper grids</td>
			<td>Ted Pella Inc.</td>
			<td>01754-F</td>
			<td>grids for negative stain</td>
		</tr>
		<tr>
			<td>Quantifoil holey carbon grid (gold, 1.2/1.3&#8201;&#956;m size/hole space, 300 mesh)</td>
			<td>Electron Microscopy Sciences</td>
			<td>Q3100AR1.3</td>
			<td>grids for Cryo-EM</td>
		</tr>
		<tr>
			<td>Vitrobot Mark III</td>
			<td>FEI</td>
			<td></td>
			<td>for preparing sample grids by liquid ethane freezing</td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td>Dura-Cyl</td>
			<td>UN1977</td>
			<td></td>
		</tr>
		<tr>
			<td>ethane gas</td>
			<td>Airgas</td>
			<td>UN1035</td>
			<td></td>
		</tr>
		<tr>
			<td>Solarus Plasma System</td>
			<td>Gatan</td>
			<td>Model 950</td>
			<td>for cleaning grids before sample freezing</td>
		</tr>
		<tr>
			<td>Tecnai Spirit electron microscope</td>
			<td>FEI</td>
			<td></td>
			<td>for negative stain EM imaging</td>
		</tr>
		<tr>
			<td>Talos Arctica electron microsocope</td>
			<td>FEI</td>
			<td></td>
			<td>for screening and low resolution imaging of Cryo-EM grids</td>
		</tr>
		<tr>
			<td>Titan Krios electron microscope</td>
			<td>FEI</td>
			<td></td>
			<td>for high-resolution Cryo-EM imaging</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gautomatch software</td>
			<td>http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/</td>
			<td></td>
			<td>to pick particles from micrographs</td>
		</tr>
		<tr>
			<td>Relion 2.1 software</td>
			<td>https://github.com/3dem/relion</td>
			<td></td>
			<td>to construct 2D and 3D classification</td>
		</tr>
		<tr>
			<td>CryoSPARC software</td>
			<td>https://cryosparc.com/</td>
			<td></td>
			<td>to generate an initial structure model</td>
		</tr>
		<tr>
			<td>Frealign software</td>
			<td>http://grigoriefflab.janelia.org/frealign</td>
			<td></td>
			<td>to refine particles</td>
		</tr>
		<tr>
			<td>Coot software</td>
			<td>https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/</td>
			<td></td>
			<td>to build a model</td>
		</tr>
		<tr>
			<td>MolProbity software</td>
			<td>http://molprobity.biochem.duke.edu/</td>
			<td></td>
			<td>to evaluate the geometries of the atomic model</td>
		</tr>
		<tr>
			<td>SerialEM software</td>
			<td>http://bio3d.colorado.edu/SerialEM/</td>
			<td></td>
			<td>for automated serial image stack acquisition</td>
		</tr>
		<tr>
			<td>MortionCor2 software</td>
			<td>http://msg.ucsf.edu/em/software/motioncor2.html</td>
			<td></td>
			<td>for motion correction of summed movie stacks</td>
		</tr>
		<tr>
			<td>GCTF software</td>
			<td>https://www.mrc-lmb.cam.ac.uk/kzhang/Gctf/</td>
			<td></td>
			<td>for measuring defocus values in movie stacks</td>
		</tr>
		<tr>
			<td>Phenix.real_space_refine software</td>
			<td>https://www.phenix-online.org/documentation/reference/real_space_refine.html</td>
			<td></td>
			<td>for real space refinement of the initial 3D model</td>
		</tr>
	</tbody>
</table>

expression and purification of the human lipid-sensitive cation channel trpc3 for structural determination by single-particle cryo-electron microscopy

典型 trp 亚家族的瞬态受体电位通道 (trpc) 是非选择性阳离子通道, 在钙稳态中发挥着至关重要的作用, 特别是在商店操作的钙进入, 这对于保持钙的适当功能至关重要。突触囊泡释放和细胞内信号通路。因此, trpc 渠道与各种人类疾病有关, 包括心血管疾病, 如心肌肥大, 神经退行性疾病, 如帕金森病, 和神经疾病, 如脊柱小脑共济失调。因此, trpc 通道是人类疾病中潜在的药理靶点。然而, 在这些通道中门控的分子机制仍不清楚。在结构测定研究中, 特别是对于哺乳动物膜蛋白, 如 trpc 离子通道, 获得大量稳定、均匀和纯化蛋白的困难一直是一个限制因素。在这里, 我们提出了一个协议, 大规模表达哺乳动物离子通道膜蛋白使用一个修改后的巴库氏病毒基因转移系统和纯化这些蛋白质的亲和力和大小排除色谱。我们进一步提出了一个协议, 收集单粒子冷冻电子显微镜图像从纯化的蛋白质, 并使用这些图像来确定蛋白质的结构。结构确定是了解离子通道中门控和功能机制的有力方法。

图 1a 显示了 htrpc3 表达和纯化协议的示意图。图 1b 显示了 htrpc3 带理想白色菌落的图像, 类似于为 bacmacmt dna 纯化选择的基板。我们发现, 48小时是理想的明确蓝甲染色, 同时保持孤立的殖民地的存在。Sf9 荧光显示, htrpc3 p2 病毒的峰值产量是在 sf9 昆虫细胞感染 4 d 后出现的 (图 1c)。
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Watch this Scientific Journal Video about Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy at JoVE

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

该协议描述了用低温电子显微镜测定离子通道结构的技术, 包括用于以最小的努力和毒性在哺乳动物细胞中有效表达基因的杆菌病毒系统, 蛋白质提取、纯化、和质量检查, 样品网格的制备和筛选, 以及数据的收集和处理。

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

该协议使我们能够解决人类TRPC3的结构，一个重要但研究不足的离子通道家族的成员。该技术的主要优点是，在结构和功能研究中，可以应用高效来表达和纯化各种蛋白质。该方法可提供有效的蛋白质表达和纯化系统，用于研究蛋白质生物化学和生物物理，并可应用于多种离子通道和其他膜蛋白。首先，通过查看病毒生成培养的样本中的病毒的 GFP 荧光来检查相对病毒表达。在一个困惑的底部 Erlenmeyer 培养瓶足够大小， 准备一个 HEK293 哺乳动物细胞悬浮培养物的理想的体积， 浓度为 350 万至 380 万细胞每毫升在表达介质中， 辅以 1% 无菌 FBS.加入8%的P2病毒库存溶液，并在37摄氏度和135 RPM的轨道摇床中孵育。感染后12至18小时加入10毫摩尔钠，在30摄氏度下孵育，达到最佳蛋白质表达所需的时间。在此之后，离心机在2，880倍重力20分钟收获细胞。每升收获的细胞中大约100毫升TBS中清洗和重新暂停细胞。在重力2，880倍下再次离心20分钟，并收集细胞颗粒。还要收集小，一毫升细胞颗粒收获在不同的时间点，溶解两个小时在四摄氏度与搅拌在不同的洗涤剂和/或添加剂的存在。通过超离心在23.5万倍重力下和在4摄氏度下10分钟，通过超离心来澄清这些小全细胞溶解样品。然后，将它们作为 30 微升样品运行在 SEC 色谱柱上，以确定表达式的最佳时间和最佳溶解条件。每升收获的细胞中，将颗粒解冻100毫升缓冲液。一旦细胞解冻，移液器或搅拌它们，以确保溶液是均匀的。让细胞在摄氏四度时在烧杯中溶解，在冰中浸入两个小时，同时被搅拌棒搅拌。接下来，在重力23.5万倍和摄氏4度的超中心离，清除任何细胞碎片，一小时。HPLC 在 SCC 列上运行 30 微升的上清液样本，以验证蛋白质数量，并通过 GFB 信号输出可视化目标蛋白质。将含有溶解蛋白的钴亲和树脂结合上清液应用到重力柱上，并收集流经。在 SCC 柱上运行流经的 30 微升样本，以验证蛋白质与树脂是否结合。然后，用10列缓冲液清洗树脂。在 SCC 柱上运行 30 微升洗涤样本，以检查蛋白质损失。使用缓冲液，对树脂结合的人类TRPC3进行成型。运行在 SSC 柱上稀释 1 到 100 的洗液的 90 微升样品，以检查蛋白质是否已被洗洗，并验证与目标蛋白质大小对应的位置是否存在 GFP 信号。以 1 到 20 摩尔的比例添加血栓，并将 EDTA 的 10 毫摩尔添加到洗洗样品中。在四摄氏度下孵育三小时。在此之后，将 Eluant 转移到 15 毫升离心过滤管。在重力2，800倍和4摄氏度的增量下旋转管，将水液浓缩到500微升或更少。在旋转之间上下移液蛋白质溶液，以重新暂停蛋白质，防止过度浓缩。将浓缩物加载到缓冲区中的 SSC 列上。运行快速蛋白质液相色谱，收集300微升分数。然后，将包含 TRPC3 四面体的峰值分数组合为 UV 吸光信号可视化。并再次浓缩到每毫升至少5毫克的最终浓度。人类TRPC3是全细胞溶解在各种洗涤剂中，临界微摩尔浓度为0.01至20微摩尔，并在含有缓冲液的DDM/CHS中运行。虽然 DDM/CHS 显示人类 TRPC3 的最高溶解度，但峰值位置看起来太大，不能成为四元人类 TRPC3。整个细胞溶解后，细胞溶解碎片被去除，溶解蛋白被加载到SSC柱上，在含有缓冲液的DDM/CHS洗涤剂中运行在HPLC上，以比较与TRPM4控制相比的不同条件下人类TRPC3的绝对溶解度和峰值体积。所有不同的洗涤剂溶解TRPC3样品都显示峰值位置在11.9毫升左右，这很可能是太大的，因为人类TRPC3的四元形式分子量小于阳性人体控制TRPM4。然后测试两种不同的溶解和运行包含 DDM/CHS 和 digitonin 的缓冲区。蛋白质在含有数字素的缓冲液中运行，相对于正对照，在合理位置产生最高峰值。虽然使用25毫升细胞进行小规模纯化，显示了几个宽阔的峰值，但蛋白质通过负染色显示单四边形人类TRPC3通道在2D分类中的特征。然后根据人类TRPC3的生理特性对添加剂进行筛选。EDTA 对将人类 TRPC3 稳定在完整的四元峰值位置具有显著影响，显著增加了低温-EM 显微图中的粒子数量，并降低了背景中的噪声。最终结果的质量取决于对 HPLC 上的 FSCC 配置文件进行良好的故障排除。在快速完成所有净化步骤时，理想情况下，在一天。结构测定、抗体生成和功能研究，如结合测定或电生理学，可以在此过程后进行。该技术可以而且已经用于研究其他离子通道和蛋白质系列，并生产纯化蛋白质，用于结构测定以外的应用。应采取适当的预防措施，管理细胞培养生物危害，如在层流罩中工作，穿着防护服，以及妥善处理废物。

Research

JoVE Journal

Biochemistry

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

科研

生物化学

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

快速网格准备时间解决低温电子显微镜

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

单颗粒冷冻电子显微镜：从样品到结构

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

用于单颗粒低温电子显微镜的生物标本的手动印迹和切入式冷冻

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

微图案透射电子显微镜网格，用于在全细胞冷冻电子断层扫描工作流程中指导细胞定位

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

强大的单颗粒冷冻电子显微镜 （cryo-EM） 处理工作流程，具有冷冻 SPARC、RELION 和 Scipion

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination

制备与DNA修复因子络合的核小体核心颗粒用于冷冻电镜结构测定

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of ...

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon

使用Leginon进行载物台倾斜的单颗粒冷冻电镜数据收集

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure ...

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

从哺乳动物细胞中提取修饰的微管以通过冷冻电子显微镜研究微管 - 蛋白质复合物

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the ...