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

Funding for this research was provided by: National Key Research and Development Program of China (2017YFD0201400, 2017YFD0201403), National Nature Science Foundation of China (31320103922, 31230061), and Project of National Basic Research (973) Program of China (2015CB856500, 2015CB856504). We are grateful to the staff on the beamline BL17U1 at Shanghai Synchrotron Radiation Facility (SSRF).

1. Gene Cloning, Protein Expression, and Purification
<ol>
	<li>PCR amplify DNA corresponding to protein of interest (residues 1-205 of DBM RyR, Genbank acc. no. AFW97408) and clone into pET-28a-HMT vector by Ligation-Independent Cloning (LIC)25. This vector contains a histidine tag, MBP tag and a TEV protease cleavage site at the N-terminus15.
	<ol>
		<li>Design LIC primers for amplification of target gene with LIC-compatible 5&#8217; extensions: 
	...

<ol>
	<li>Giannini, G., Sorrentino, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+structure+and+tissue+distribution+of+ryanodine+receptors+calcium+channels.">Molecular structure and tissue distribution of ryanodine receptors calcium channels.</a> Medicinal Research Reviews. 15 (4), 313-323 (1995).</li><li>Takeshima, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+a+gene+for+a+ryanodine+receptor/calcium+release+channel+in+Drosophila+melanogaster.">Isolation and characterization of a gene for a ryanodine receptor/calcium release channel in Drosophila melanogaster.</a> FEBS Letters. 337 (1), 81-87 (1994).</li><li>Sattelle, D. B., Cordova, D., Cheek, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+ryanodine+receptors:+molecular+targets+for+novel+pest+control+chemicals.">Insect ryanodine receptors: molecular targets for novel pest control chemicals.</a> Invertebrate Neuroscience. 8 (3), 107-119 (2008).</li><li>Steinbach, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+spread,+genetics+and+functional+characteristics+of+ryanodine+receptor+based+target-site+resistance+to+diamide+insecticides+in+diamondback+moth,+Plutella+xylostella.">Geographic spread, genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth, Plutella xylostella.</a> Insect Biochemistry and Molecular Biology. 63, 14-22 (2015).</li><li>Wang, X., Khakame, S. K., Ye, C., Yang, Y., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+field-evolved+resistance+to+chlorantraniliprole+in+the+diamondback+moth,+Plutella+xylostella,+from+China.">Characterisation of field-evolved resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella, from China.</a> Pest Management Science. 69 (5), 661-665 (2013).</li><li>Liu, X., Wang, H. Y., Ning, Y. B., Qiao, K., Wang, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+Selection+and+Characterization+of+Chlorantraniliprole+Resistance+in+Plutella+xylostella+(Lepidoptera:+Plutellidae).">Resistance Selection and Characterization of Chlorantraniliprole Resistance in Plutella xylostella (Lepidoptera: Plutellidae).</a> Journal of Economic Entomology. 108 (4), 1978-1985 (2015).</li><li>Guo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+a+point+mutation+in+the+ryanodine+receptor+of+Plutella+xylostella+(L.)+associated+with+resistance+to+chlorantraniliprole.">Functional analysis of a point mutation in the ryanodine receptor of Plutella xylostella (L.) associated with resistance to chlorantraniliprole.</a> Pest Management Science. 70 (7), 1083-1089 (2014).</li><li>Troczka, 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=Resistance+to+diamide+insecticides+in+diamondback+moth,+Plutella+xylostella+(Lepidoptera:+Plutellidae)+is+associated+with+a+mutation+in+the+membrane-spanning+domain+of+the+ryanodine+receptor.">Resistance to diamide insecticides in diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) is associated with a mutation in the membrane-spanning domain of the ryanodine receptor.</a> Insect Biochemistry and Molecular Biology. 42 (11), 873-880 (2012).</li><li>Roditakis, 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=Ryanodine+receptor+point+mutations+confer+diamide+insecticide+resistance+in+tomato+leafminer,+Tuta+absoluta+(Lepidoptera:+Gelechiidae).">Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).</a> Insect Biochemistry and Molecular Biology. 80, 11-20 (2017).</li><li>Borko, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+human+RyR2+N-terminal+region+involved+in+cardiac+arrhythmias.">Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias.</a> Acta Crystallographica Section D. 70 (Pt 11), 2897-2912 (2014).</li><li>Sharma, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+determination+of+the+phosphorylation+domain+of+the+ryanodine+receptor.">Structural determination of the phosphorylation domain of the ryanodine receptor.</a> FEBS Journal. 279 (20), 3952-3964 (2012).</li><li>Kimlicka, L., Lau, K., Tung, C. C., Van Petegem, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease+mutations+in+the+ryanodine+receptor+N-terminal+region+couple+to+a+mobile+intersubunit+interface.">Disease mutations in the ryanodine receptor N-terminal region couple to a mobile intersubunit interface.</a> Nature Communications. 4, 1506 (2013).</li><li>Lau, K., Van Petegem, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structures+of+wild+type+and+disease+mutant+forms+of+the+ryanodine+receptor+SPRY2+domain.">Crystal structures of wild type and disease mutant forms of the ryanodine receptor SPRY2 domain.</a> Nature Communications. 5, 5397 (2014).</li><li>Amador, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+type+I+ryanodine+receptor+amino-terminal+beta-trefoil+domain+reveals+a+disease-associated+mutation+&amp;#34;hot+spot&amp;#34;+loop.">Crystal structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation &amp;#34;hot spot&amp;#34; loop.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (27), 11040-11044 (2009).</li><li>Lobo, P. A., Van Petegem, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structures+of+the+N-terminal+domains+of+cardiac+and+skeletal+muscle+ryanodine+receptors:+insights+into+disease+mutations.">Crystal structures of the N-terminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations.</a> Structure. 17 (11), 1505-1514 (2009).</li><li>des Georges, 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=Structural+Basis+for+Gating+and+Activation+of+RyR1.">Structural Basis for Gating and Activation of RyR1.</a> Cell. 167 (1), 145-157 (2016).</li><li>Efremov, R. G., Leitner, A., Aebersold, R., Raunser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+conformational+switch+mechanism+of+the+ryanodine+receptor.">Architecture and conformational switch mechanism of the ryanodine receptor.</a> Nature. 517 (7532), 39-43 (2015).</li><li>Peng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+gating+mechanism+of+the+type+2+ryanodine+receptor+RyR2.">Structural basis for the gating mechanism of the type 2 ryanodine receptor RyR2.</a> Science. 354 (6310), (2016).</li><li>Wei, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+Ca2+-activated+long-range+allosteric+channel+gating+of+RyR1.">Structural insights into Ca2+-activated long-range allosteric channel gating of RyR1.</a> Cell Research. 26 (9), 977-994 (2016).</li><li>Yan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+rabbit+ryanodine+receptor+RyR1+at+near-atomic+resolution.">Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution.</a> Nature. 517 (7532), 50-55 (2015).</li><li>Zalk, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+mammalian+ryanodine+receptor.">Structure of a mammalian ryanodine receptor.</a> Nature. 517 (7532), 44-49 (2015).</li><li>Furlong, M. J., Wright, D. J., Dosdall, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diamondback+moth+ecology+and+management:+problems,+progress,+and+prospects.">Diamondback moth ecology and management: problems, progress, and prospects.</a> Annual Review of Entomology. 58, 517-541 (2013).</li><li>Amador, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+type+I+ryanodine+receptor+amino-terminal+beta-trefoil+domain+reveals+a+disease-associated+mutation+&amp;#34;hot+spot&amp;#34;+loop.">Crystal structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation &amp;#34;hot spot&amp;#34; loop.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (27), 11040-11044 (2009).</li><li>Lobo, P. A., Van Petegem, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structures+of+the+N-Terminal+Domains+of+Cardiac+and+Skeletal+Muscle+Ryanodine+Receptors:+Insights+into+Disease+Mutations.">Crystal Structures of the N-Terminal Domains of Cardiac and Skeletal Muscle Ryanodine Receptors: Insights into Disease Mutations.</a> Structure. 17 (11), 1505-1514 (2009).</li><li>Aslanidis, C., de Jong, P. 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C., 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=Iterative+model+building,+structure+refinement+and+density+modification+with+the+PHENIX+AutoBuild+wizard.">Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard.</a> Acta Crystallographica Section D. 64 (Pt 1), 61-69 (2008).</li><li>Emsley, P., 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=Coot:+model-building+tools+for+molecular+graphics.">Coot: model-building tools for molecular graphics.</a> Acta Crystallographica Section D. 60, 2126-2132 (2004).</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. 68 (Pt 4), 352-367 (2012).</li><li>Lin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+ryanodine+receptor+N-terminal+domain+from+Plutella+xylostella+reveals+two+potential+species-specific+insecticide-targeting+sites.">Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites.</a> Insect Biochemistry and Molecular Biology. 92, 73-83 (2018).</li><li>Qi, S., Casida, J. 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In this paper, we describe the procedure to recombinantly express, purify, crystallize and determine the structure of DBM RyR NTD. For crystallization, a crucial requirement is to obtain proteins with high solubility, purity and homogeneity. In our protocol, we chose to use pET-28a-HMT vector as it contains a hexahistidine tag and MBP tag, both of which could be utilized for purification to obtain a higher fold purity. Additionally, the MBP tag aids in the solubility of the target protein. We purified the protein by five...

Ryanodine receptors (RyRs) are specific ion channels, which mediate the permeation of Ca2+ ions across the sarcoplasmic reticulum (SR) membranes in muscle cells. Therefore, they play an important role in the excitation contraction coupling process. In its functional form, RyR assembles as a homo-tetramer with a molecular mass of &#62;2 MDa, with each subunit comprising of ~5000 amino acid residues. In mammals, there are three isoforms: RyR1- skeletal muscle type, RyR2- cardiac muscle type and RyR3- ubiquitously expressed in different tissues1.
In insects there is only one type of RyR, which is expressed in muscular and nervous tissue2. Insect RyR is more similar to mammalian RyR2 with a sequence identity of about 47%3. Diamide insecticides targeting RyR of Lepidoptera and Coleoptera have been developed and marketed by major companies like Bayer (flubendiamide), DuPont (chlorantraniliprole) and Syngenta (cyantraniliprole). Since its relatively recent launch, diamide insecticides have become one of the fastest growing class of insecticides. Currently, the sales of these three insecticides annually have crossed 2 billion U.S. dollars with a growth rate of more than 50% since 2009 (Agranova).
Recent studies have reported the development of resistance in insects after a few generations of usage of these insecticides4,5,6,7,8. The resistance mutations in the transmembrane domain of RyRs from the diamondback moth (DBM), Plutella xylostella (G4946E, I4790M) and the corresponding positions in tomato leafminer, Tuta absoluta (G4903E, I4746M) show that the region might be involved in diamide insecticide binding as this region is known to be critical for gating of the channel4,8,9. Despite extensive research in this area, the exact molecular mechanisms of diamide insecticides remain elusive. Moreover, it is unclear whether the resistance mutations affect the interactions with diamides directly or allosterically.
Earlier studies have reported the structure of several RyR domains from mammalian species and the structure of full-length mammalian RyR1 and RyR2 by x-ray crystallography and cryo-electron microscopy, respectively10,11,12,13,14,15,16,17,18,19,20,21. But so far, no structure of insect RyR has been reported, which prohibits us from understanding the molecular intricacies of the receptor function as well as the molecular mechanisms of insecticide action and development of insecticide resistance.
In this manuscript, we present a generalized protocol for the structural characterization of N-terminal &#946;-trefoil domain of ryanodine receptor from the diamondback moth, a destructive pest infecting cruciferous crops worldwide22. The construct was designed according to the published rabbit RyR1 NTD crystal structures23,24and the cryo-EM structural models16,17,18,19,20,21. This is the first high-resolution structure reported for insect RyR, which reveals the mechanism for channel gating and provides an important template for the development of species-specific insecticides using structure-based drug design. For structure elucidation, we employed x-ray crystallography, which is considered as the &#39;gold standard&#39; for protein structure determination at near atomic resolution. Although the crystallization process is unpredictable and labor intensive, this step-by-step protocol will help researchers to express, purify and characterize other domains of insect RyR or any other proteins in general.

<table border="0" cellpadding="0" cellspacing="0" width="1450">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pET-28a-HMT vector</td>
			<td style="width:164px;"></td>
			<td style="width:129px;"></td>
			<td style="width:941px;">This modified pET vector contains a hexahistidine tag, an MBP fusion protein and a TEV protease cleavage site at the N-terminus (Lobo and Van Petegem, 2009)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E. coli BL21 (DE3) strain</td>
			<td style="width:164px;">Novagen</td>
			<td>69450-3CN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HisTrapHP column (5 mL)</td>
			<td>GE Healthcare</td>
			<td>45-000-325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amylose resin column</td>
			<td>New England Biolabs</td>
			<td style="width:129px;">E8021S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q Sepharose high-performance column&#160;</td>
			<td>GE Healthcare</td>
			<td>17-1154-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon concentrators (10 kDa MWCO)</td>
			<td>Millipore</td>
			<td>UFC901008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superdex 200 26/600 gel-filtration column&#160;</td>
			<td>GE Healthcare</td>
			<td>28-9893-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automated liquid handling robotic system&#160;</td>
			<td>Art Robbins Instruments</td>
			<td style="width:129px;">Gryphon</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 Well CrystalQuick</td>
			<td>Greiner bio-one</td>
			<td>82050-494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Uni-Puck</td>
			<td>Molecular Dimensions</td>
			<td style="width:129px;">MD7-601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounted CryoLoop - 20 micron</td>
			<td>Hampton Research</td>
			<td style="width:129px;">HR4-955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CryoWand</td>
			<td>Molecular Dimensions</td>
			<td style="width:129px;">MD7-411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puck dewar loading tool</td>
			<td style="width:164px;">Molecular Dimensions</td>
			<td>MD7-607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nano drop</td>
			<td style="width:164px;">Thermo Scientific</td>
			<td style="width:129px;">NanoDrop One</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Crystal incubator</td>
			<td style="width:164px;">Molecular Dimensions</td>
			<td style="width:129px;">MD5-605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">X-Ray diffractor</td>
			<td style="width:164px;">Rigaku</td>
			<td style="width:129px;">FRX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PCR machine</td>
			<td style="width:164px;">Eppendorf</td>
			<td style="width:129px;">Nexus GX2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plasmid mini-prep kit</td>
			<td style="width:164px;">Qiagen</td>
			<td align="right" style="width:129px;">27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel extraction kit</td>
			<td style="width:164px;">Qiagen</td>
			<td align="right" style="width:129px;">28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SspI restriction endonuclease</td>
			<td style="width:164px;">NEB</td>
			<td style="width:129px;">R0132S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 DNA polymerase</td>
			<td style="width:164px;">Novagen</td>
			<td align="right" style="width:129px;">2868713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kanamycin</td>
			<td style="width:164px;">Scientific Chemical</td>
			<td align="right" style="width:129px;">25389940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IPTG</td>
			<td style="width:164px;">Genview</td>
			<td align="right" style="width:129px;">367931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:164px;">Genview</td>
			<td align="right" style="width:129px;">7365459</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol</td>
			<td style="width:164px;">Genview</td>
			<td align="right" style="width:129px;">60242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:164px;">Thermo Scientific</td>
			<td style="width:129px;">Sorvall LYNX 6000&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonnicator</td>
			<td style="width:164px;">Scientz</td>
			<td style="width:129px;">II-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protein purification system</td>
			<td style="width:164px;">GE Healthcare</td>
			<td style="width:129px;">Akta Pure</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light microscope</td>
			<td style="width:164px;">Nikon</td>
			<td style="width:129px;">SMZ745</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IzIt crystal dye</td>
			<td style="width:164px;">Hampton Research</td>
			<td>HR4-710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrophoresis unit</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:129px;">1658005EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaker Incubator</td>
			<td style="width:164px;">Zhicheng</td>
			<td style="width:129px;">ZWYR-D2401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Index crystal screen</td>
			<td style="width:164px;">Hampton Research</td>
			<td style="width:129px;">HR2-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Structure crystal screen</td>
			<td style="width:164px;">Molecular Dimensions</td>
			<td style="width:129px;">MD1-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ProPlex crystal screen</td>
			<td style="width:164px;">Molecular Dimensions</td>
			<td style="width:129px;">MD1-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PACT premier crystal screen</td>
			<td style="width:164px;">Molecular Dimensions</td>
			<td style="width:129px;">MD1-29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">JCSG-plus crystal screen</td>
			<td style="width:164px;">Molecular Dimensions</td>
			<td style="width:129px;">MD1-37</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Purification
The N-terminal domain of DBM RyR was expressed as a fusion protein with a hexahistidine tag, a MBP tag&#160;and a TEV protease cleavage site. We followed a five-step purification strategy to obtain a highly pure protein, suitable for crystallization purpose. At first, the fusion protein was purified from the soluble fraction of cell lysate by Ni-NTA column (HisTrap HP). Next, the fusion protein was subjected to TEV pro...

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

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

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

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

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

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