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 ...

Solving the structure of the ryanodine receptor domain can help with our understanding of the molecular mechanism of the protein function, insecticide action, and insecticide resistance development. This measure implies the use of X-ray crystallography for structure illustration, which is considered a gold standard for protein structure determination at near-atomic resolution. This high-resolution structure of the insect ryanodine receptor domain reveals the mechanism of channel gating and provides an important template for the development of species-specific insecticides using structure-based drug design approaches.Generally, individuals new to this method will struggle, because prospective protein crystallographers must be proficient in biochemistry, biophysics, computer science, and maths. Begin by amplifying the DNA corresponding to the protein of interest by polymerase chain reaction. At the end of the reaction, run the entire 50 microliters of reaction mix on a 2%agarose gel, and extract the DNA with a gel extraction kit according to standard protocols.Next, linearize the LIC vector DNA by mixing 20 microliters of the vector DNA with 6 microliters of 10X reaction buffer and 4 microliters of SspI restriction enzyme in 30 microliters of double-distilled water. Incubate this reaction mixture for three hours at 37 degrees Celsius. At the end of the incubation, run the entire reaction mix on a 1%agarose gel, followed by extraction of the linearized vector DNA with a gel extraction kit according to the manufacturer's instructions.Next, perform separate T4 DNA polymerase treatments on 5 microliters of the linearized vector DNA and the PCR amplified insert DNA, according to standard protocols. Incubate for 40 minutes at room temperature, followed by 20 minutes of enzyme heat inactivation at 75 degrees Celsius. Perform the ligation-independent cloning annealing reaction by combining 2 microliters of the T4 treated insert DNA with 2 microliters of the T4 treated LIC vector DNA for a 10 minute incubation at room temperature.To transform BL21(DE3)E. Coli cells with the recombinant plasmid, thaw 50 microliters of competent cells on ice, and add approximately 1 microliter of the annealed plasmid to the tube. Gently flick the tube two to three times to mix the cells and the DNA, and place the tube back on ice for 20 minutes.At the end of the incubation, heat-shock the cells at 42 degrees Celsius for 40 seconds, followed by two minutes back on ice. Next, add one milliliter of room-temperature LB medium to the tube, and place the cells on a 250 RPM shaker for 45 minutes at 37 degrees Celsius. At the end of the shaking incubation, plate 150 to 200 microliters of this mixture onto selection plates, and invert the plates overnight at 37 degrees Celsius.The next day, culture a single colony in 100 milliliters of 2-YT medium, supplemented with kanamycin, in a shaker incubator at 37 degrees Celsius overnight. The next morning, inoculate one liter of 2-YT medium, supplemented with kanamycin, with 10 milliliters of overnight culture, and incubate at 37 degrees Celsius with shaking until the OD600 reading reaches approximately 0.6. Then, induce the culture with IPTG to a final concentration of 0.4 millimolar, and grow the cells for five hours at 30 degrees Celsius.At the end of the incubation, harvest the cells by centrifugation, and re-suspend the pellet to a 10 grams of bacteria per 40 milliliters of lysis buffer concentration. Disrupt the cell walls by sonication at a 65%amplitude, and one second on one second off, for eight minutes. Remove the cell debris by centrifugation.Then, filter the supernatant though a 22 micrometer filter, and load the solution into a sample loop. To purify the fusion protein, inject the filtered supernatant from the sample loop into a five milliliter nickel-nitrolotriacetic column on a purification system with a linear gradient of 20 to 250 millimolar imidazole. Cleave the eluted target protein with tobacco etch virus protease, at a 1-to-50 ratio, overnight at 4 degrees Celsius, and purify the cleavage reaction mixture on an amylose resin column, and a nickel-nitrolotriacetic column, to remove the tag and the tobacco etch virus protease.Dialyze the flow-through from the nickel-nitrolotriacetic column against dialysis buffer to reduce the salt concentration, and purify the sample on an anion exchange column by a linear gradient of 20 to 500 millimolar potassium chloride in the elution buffer. Then, concentrate the protein in a centrifugal concentrator. Inject the concentrated protein into a Superdex 200 26/600 gel filtration column to check the homogeneity, and assess the purity of the protein by 15%SDS-PAGE.To prepare the protein for crystallization, concentrate the purified protein sample to 10 milligrams per milliliter in the centrifugal concentrator, and buffer exchange to crystallization buffer before 80 degree Celsius storage. To perform crystallization screening, use the sitting drop vapor diffusion method at 295 Kelvin, with several crystallization kits, and an automated liquid-handling system in a 96-well format. At the end of the drop setting, seal the 96-well crystallization plate to prevent evaporation, and to enable the equilibration of the protein drop within the reservoir buffer.Place the plates in a crystal incubator at 18 degrees Celsius. Check the plates periodically under a light microscope to monitor the crystal formation and growth. To differentiate the protein crystals from the salt crystals, add one microliter of protein crystal dye to the target drop, and observe the crystals under the microscope after one hour.The protein crystals will appear blue. To further optimize the crystals, use the positive crystallization conditions and the hanging drop vapor diffusion method in 24-well plates, followed by incubation in the crystal incubator at 18 degrees Celsius. To determine the protein structure, mount the crystals on a cryoloop under a light microscope for flash cooling in liquid nitrogen, and place the crystals in a unibuck for storage and transportation.Pre-screen the crystals in an in-house X-ray diffractor, using the manual centering function to mount and center the crystals. Then, use the X-ray diffraction software to collect the data. To index, integrate and scale the data set, using HKL-2000 suite, first, select the detector, and load the data set.Then, carry out the peak search function to find the diffraction spots. Index the spots, select the right space group, and perform peak integration. Next, scale the data set.Adjust the error model and scale again. Save the output sca file. To determine the structure using Phenix software suite, create a new project.First, run Extriage with the sca file to calculate the possible copy number of protein molecules in the asymmetric unit. Next, to solve the phase problem by molecular replacement, run Phaser, using the diffraction data file, the template structure file with high-sequence identity and structural similarity as the target protein, and the protein sequence file, to find the solution. Perform auto-build in Phenix to generate the initial model, using the output file from Phaser and the sequence file from the target protein.Manually build the structure into the modified experimental electron density using COOT, and refine using Phenix refine in iterative cycles. Validate the final model using the validation tools in Phenix. In this representative experiment, purification of the end-terminal domain of the diamondback moth ryanodine receptor protein, as demonstrated, yielded a single band at about 21 kilodaltons by STS-PAGE analysis.The elution volume from the gel filtration column confirmed the purified ryanodine receptor end-terminal domain to be a monomer. For crystallization, the most optimal conditions under which high-quality plate-shaped crystals were formed was in the presence of 1 molar HEPES of a pH of 7, and 1.6 molar ammonium sulfate. Determination of the protein structure from the diffraction data set utilizing softwares as demonstrated revealed the diamondback moth ryanodine receptor end-terminal domain covering residues 1 through 205.Proteins with large disorder, flexible regions, or with weak affinities are challenging to crystallize. In these scenarios, protein engineering strategies such as surface entropy reduction, loop truncation, and cross-linking, may increase the likelihood of obtaining better protein crystals. In addition to revealing high-resolution protein structures, X-ray crystallography may also be used to study protein-pesticide interactions, which could help with structure-based pesticide design.

crystal-structure-n-terminal-domain-ryanodine-receptor-from-plutella

Research

JoVE Journal

Biochemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

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

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

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

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

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

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

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

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

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

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

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies an invaluable tool for biomedical research. A method for the production of single domain antibodies from alpacas is reported. A protocol for immunization, blood collection, and B-cell isolation is described. The B-cells are used for the construction of an immunized library, which is used in the selection of specific single domain antibodies via panning. Putative specific single domain antibodies obtained via panning are confirmed by pull-down, ELISA, or gel-shift assays. The resulting single domain antibodies can then be used either directly or as a part of an engineered reagent. The uses of single domain antibody and single domain antibody-based regents include structural, biochemical, cellular, in vivo, and therapeutic applications. Single domain antibodies can be produced in large quantities as recombinant proteins in prokaryotic expression systems, purified, and used directly or can be engineered to contain specific markers or tags that can be used as reporters in cellular studies or in diagnostics.

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

A method for the production of single domain antibodies from alpacas, including immunization, blood collection, B-cell isolation, and selection is described.

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

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological macromolecules. Until recently, a major requirement has been the availability of relatively large, well diffracting crystals, which are often challenging to obtain. However, the advent of serial crystallography and a renaissance in multi-crystal data collection methods has meant that the availability of large crystals need no longer be a limiting factor. Here, we illustrate the use of the automated MeshAndCollect protocol, which first identifies the positions of many small crystals mounted on the same sample holder and then directs the collection from the crystals of a series of partial diffraction data sets for subsequent merging and use in structure determination. MeshAndCollect can be applied to any type of micro-crystals, even if weakly diffracting. As an example, we present here the use of the technique to solve the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean.

We present the use of the MeshAndCollect protocol to obtain a complete diffraction data set, for use in subsequent structure determination, composed of partial diffraction data sets collected from many small crystals of the fluorescent protein Cerulean.

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

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

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

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

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

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 ...