Name: expression and purification of the human lipid-sensitive cation channel trpc3 for structural determination by single-particle cryo-electron microscopy
Uploaded: 2019-01-07T14:00:00
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 Video a

We thank G. Zhao and X. Meng for the support with data collection at the David Van Andel Advanced Cryo-Electron Microscopy Suite. We appreciate the VARI High-Performance Computing team for computational support. We give our gratitude to N. Clemente, D. Dues, J. Floramo, Y. Huang, Y. Kim, C. Mueller, B. Roth, and Z. Ruan for comments that greatly improved this manuscript. We thank D. Nadziejka for editorial support for this manuscript. This work was supported by internal VARI funding.

1. Transformation of DH10&#945; Competent Cells to Produce Bacmid DNA
<ol>
	<li>Synthesize the gene of interest and subclone it into a modified version of the pEG vector containing a twin strep-tag, a His8-tag, and GFP with a thrombin cleavage site at the N terminus (pFastBacI)20.</li>
	<li>Transform competent cells by adding 5 ng of plasmid containing a desired gene in pFastBacI to 50 &#956;L of DH10&#945; cells in a 1.5 mL tube and incubate for 10 min on ice. Heat shock the cells for 45 s at 4...

<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>

Structural determination of proteins by cryo-EM has revolutionized the field of structural biology in the past few years, thanks to the development of new cameras and algorithms that significantly speeds up the structure determination of proteins that do not readily crystalize, particularly membrane proteins. Despite all of the recent advances in the cryo-EM technique, the preparation of purified proteins sufficient in quality and quantity to facilitate high-quality imaging often remains time-consuming, costly, and chall...

Calcium is involved in most cellular processes including signaling cascades, transcription control, neurotransmitter release, and hormone molecule synthesis1,2,3. The homeostatic maintenance of cytosolic free&#160;calcium is crucial to the health and function of cells. One of the major mechanisms of intracellular calcium homeostasis is store-operated calcium entry (SOCE), a process in which depletion of calcium stored in the endoplasmic reticulum (ER) triggers the opening of ion channels on the plasma membrane to facilitate the replenishment of ER calcium, which can then be used in further signaling4,5,6. Transient receptor potential channels (TRPCs), which are calcium-permeable channels belonging to the TRP superfamily, have been identified as a major participant in SOCE7,8,9 .
Among the seven members in the TRPC family, TRPC3, TRPC6, and TRPC7 form a homologue subgroup, and they are unique in the ability to be activated by the lipid secondary messenger diacylglycerol (DAG), a degradation product of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2)10,11. TRPC3 is highly expressed in smooth muscle and in the cerebral and cerebellar regions of the brain, where it plays essential roles in calcium signaling that impact&#160;neurotransmission and neurogenesis12,13. Dysfunction of TRPC3 has been linked to central nervous system disorders, cardiovascular disorders, and certain cancers such as ovarian adenocarcinoma14,15,16. Therefore, TRPC3 holds promise as a pharmaceutical target for treatment of these diseases. The development of specifically targeted drugs acting on TRPC3 has been limited by a lack of understanding of its molecular activation mechanisms, including lipid binding sites17,18. We have reported the first atomic-resolution structure of the human TRPC3 channel (hTRPC3) and its two lipid binding sites in a closed state, providing important insights into these mechanisms19.
The key factor for determining the structure of a membrane protein at high resolution is to obtain protein of high quality. The corresponding screening of expression and purification conditions necessary to obtain high quality protein can be a time-consuming and costly endeavor. Here we present a protocol describing in detail how we identify the optimal conditions for the expression and purification of hTRPC3, which behaved poorly in our initial screening. We present several key points on how to troubleshoot and optimize the protein behavior, which lay a solid foundation for our cryo-electron microscopy (cryo-EM) studies. We use a modified baculoviral generating vector (pEG), developed by Gouaux and colleagues, which is optimized for screening assays and efficient generation of baculovirus in mammalian cells20. This expression method is appropriate for rapid and cost-effective overexpression of proteins in the mammalian cell membrane. We combine the use of this vector with a fluorescence-detection size-exclusion chromatography-based (FSEC) prescreening method21. This method uses a green fluorescent protein (GFP) tag fused to the construct of interest and improves visualization of the target protein in small, whole-cell solubilized samples. This allows for screening of protein stability in the presence of different detergents and additives, with thermostabilizing mutations, and allows the use of a small number of cells from small-scale transient transfection. In this way, a multitude of conditions can be rapidly screened before moving to a large-scale protein purification. Following expression, screening, and purification, we present a protocol for obtaining and processing images from cryo-EM to generate a de novo structural determination of the protein. We believe that the approaches described here will serve as a generalizable protocol for structural studies of TRP channel receptors and other membrane proteins.

<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

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

A schematic overview of the protocol for expression and purification of hTRPC3 is shown in Figure 1A. An image of the hTRPC3 bacmid plate with ideal white colonies, similar to the one selected for bacmid DNA purification, is shown in Figure 1B. We found that 48 h is ideal for clear Bluo-gal staining while maintaining the presence of isolated colonies. Peak production of P2 virus for hTRPC3, as visualized by GFP fluorescence, was ...

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 Video a

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

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

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

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

Research

JoVE Journal

Biochemistry

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

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE Video (Video) | JoVE

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

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip Video (Video) | JoVE

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy Video (Video) | JoVE

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

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography Video (Video) | JoVE

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Single Particle Cryo-Electron Microscopy: From Sample to Structure Video (Video) | JoVE

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

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy Video (Video) | JoVE

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

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows Video (Video) | JoVE

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

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Purification of Endogenous Drosophila Transient Receptor Potential Channels Video (Video) | JoVE

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...

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 Video (Video) | JoVE

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

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination Video (Video) | JoVE

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