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.

<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. 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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. 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The authors have nothing to disclose.

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 at JoVE

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

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.

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

expression-purification-human-lipid-sensitive-cation-channel-trpc3

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

9.4K Views.  Van Andel Institute. 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.

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

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

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

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

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

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

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

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

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

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

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

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

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

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

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

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

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method for structural biologists to determine high-resolution structures of a wide variety of macromolecules. Multiple software suites are available to new and expert users for image processing and structure calculation, which streamline the same basic workflow: movies acquired by the microscope detectors undergo correction for beam-induced motion and contrast transfer function (CTF) estimation. Next, particle images are selected and extracted from averaged movie frames for iterative 2D and 3D classification, followed by 3D reconstruction, refinement, and validation. Because various software packages employ different algorithms and require varying levels of expertise to operate, the 3D maps they generate often differ in quality and resolution. Thus, users regularly transfer data between a variety of programs for optimal results. This paper provides a guide for users to navigate a workflow across the popular software packages: cryoSPARC v3, RELION-3, and Scipion 3 to obtain a near-atomic resolution structure of the adeno-associated virus (AAV). We first detail an image processing pipeline with cryoSPARC v3, as its efficient algorithms and easy-to-use GUI allow users to quickly arrive at a 3D map. In the next step, we use PyEM and in-house scripts to convert and transfer particle coordinates from the best quality 3D reconstruction obtained in cryoSPARC v3 to RELION-3 and Scipion 3 and recalculate 3D maps. Finally, we outline steps for further refinement and validation of the resultant structures by integrating algorithms from RELION-3 and Scipion 3. In this article, we describe how to effectively utilize three processing platforms to create a single and robust workflow applicable to a variety of data sets for high-resolution structure determination.

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

This article describes how to effectively utilize three cryo-EM processing platforms, i.e., cryoSPARC v3, RELION-3, and Scipion 3, to create a single and robust workflow applicable to a variety of single-particle data sets for high-resolution structure determination.

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

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase &#946; and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

This protocol outlines in detail the preparation of nucleosomal complexes using two methods of sample preparation for freezing TEM grids.

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

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure determination by SPA relies upon obtaining multiple distinct views of a macromolecular object vitrified within a thin layer of ice. Ideally, a collection of uniformly distributed random projection orientations would amount to all possible views of the object, giving rise to reconstructions characterized by isotropic directional resolution. However, in reality, many samples suffer from preferentially oriented particles adhering to the air-water interface. This leads to non-uniform angular orientation distributions in the dataset and inhomogeneous Fourier-space sampling in the reconstruction, translating into maps characterized by anisotropic resolution. Tilting the specimen stage provides a generalizable solution to overcoming resolution anisotropy by virtue of improving the uniformity of orientation distributions, and thus the isotropy of Fourier space sampling. The present protocol describes a tilted-stage automated data collection strategy using Leginon, a software for automated image acquisition. The procedure is simple to implement, does not require any additional equipment or software, and is compatible with most standard transmission electron microscopes (TEMs) used for imaging biological macromolecules.

The present protocol describes a generalized and easy-to-implement scheme for tilted single-particle data collection in cryo-EM experiments. Such a procedure is especially useful for obtaining a high-quality EM map for samples suffering from preferential orientation bias due to adherence to the air-water interface.

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

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

Summary

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Chapters in this video

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

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

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Single Particle Cryo-Electron Microscopy: From Sample to Structure

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

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

Purification of Endogenous Drosophila Transient Receptor Potential Channels

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

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

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