Name: combining wet and dry lab techniques to guide the crystallization of large coiled-coil containing proteins
Uploaded: 2017-01-06T10:30:00
Description: Watch this Scientific Journal Video about Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins at JoVE.com

This work was supported by grant NIH R01 GM097204 (APV and JDH). Funding for JHM was supplied by an HHMI Undergraduate Research Summer Fellowship.

NOTE: Un diagramme du cadre conceptuel ou flux de travail est décrit dans la figure 1 pour référence. Le protocole peut être décomposé en quatre étapes: calcul des prédictions basées ou séquence, expression de protéines et de purification, caractérisation biochimique, et de cristallisation. Les exemples ci analysent les domaines Shroom SD2 et / ou complexes Shroom-Rock, mais ils peuvent être utilisés avec toute protéine. 1. Utilisez Établi outils Web pour gén?...

<ol>
	<li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+expression+clones+and+auto-induction+for+protein+production+in+E.+coli.">Stable expression clones and auto-induction for protein production in E. coli.</a> Methods Mol Biol. 1091, 17-32 (2014).</li><li>Studier, F. W., Moffatt, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bacteriophage+T7+RNA+polymerase+to+direct+selective+high-level+expression+of+cloned+genes.">Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.</a> J Mol Biol. 189 (1), 113-130 (1986).</li><li>Chapman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification:+pure+but+not+simple.">Protein purification: pure but not simple.</a> Nature. 434 (7034), 795-798 (2005).</li><li>Gibson, D. G., 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=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Lupas, A., Van Dyke, M., Stock, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+coiled+coils+from+protein+sequences.">Predicting coiled coils from protein sequences.</a> Science. 252 (5009), 1162-1164 (1991).</li><li>Offer, G., Hicks, M. R., Woolfson, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+Crick+equations+for+modeling+noncanonical+coiled+coils.">Generalized Crick equations for modeling noncanonical coiled coils.</a> J Struct Biol. 137 (1-2), 41-53 (2002).</li><li>Lupas, A. N., Gruber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+alpha-helical+coiled+coils.">The structure of alpha-helical coiled coils.</a> Adv Protein Chem. 70, 37-78 (2005).</li><li>Hernandez Alvarez, 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=A+new+expression+system+for+protein+crystallization+using+trimeric+coiled-coil+adaptors.">A new expression system for protein crystallization using trimeric coiled-coil adaptors.</a> Protein Eng Des Sel. 21 (1), 11-18 (2008).</li><li>Slabinski, 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=The+challenge+of+protein+structure+determination--lessons+from+structural+genomics.">The challenge of protein structure determination--lessons from structural genomics.</a> Protein Sci. 16 (11), 2472-2482 (2007).</li><li>Slabinski, 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=XtalPred:+a+web+server+for+prediction+of+protein+crystallizability.">XtalPred: a web server for prediction of protein crystallizability.</a> Bioinformatics. 23 (24), 3403-3405 (2007).</li><li>Haigo, S. L., Hildebrand, J. D., Harland, R. M., Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shroom+induces+apical+constriction+and+is+required+for+hingepoint+formation+during+neural+tube+closure.">Shroom induces apical constriction and is required for hingepoint formation during neural tube closure.</a> Curr Biol. 13 (24), 2125-2137 (2003).</li><li>Hildebrand, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shroom+regulates+epithelial+cell+shape+via+the+apical+positioning+of+an+actomyosin+network.">Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network.</a> J Cell Sci. 118 (Pt 22), 5191-5203 (2005).</li><li>Dietz, M. L., Bernaciak, T. M., Vendetti, F., Kielec, J. M., Hildebrand, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+actin-dependent+localization+modulates+the+evolutionarily+conserved+activity+of+Shroom+family+proteins.">Differential actin-dependent localization modulates the evolutionarily conserved activity of Shroom family proteins.</a> J Biol Chem. 281 (29), 20542-20554 (2006).</li><li>Bolinger, C., Zasadil, L., Rizaldy, R., Hildebrand, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+isoforms+of+drosophila+shroom+define+spatial+requirements+for+the+induction+of+apical+constriction.">Specific isoforms of drosophila shroom define spatial requirements for the induction of apical constriction.</a> Dev Dyn. 239 (7), 2078-2093 (2010).</li><li>Farber, M. J., Rizaldy, R., Hildebrand, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shroom2+regulates+contractility+to+control+endothelial+morphogenesis.">Shroom2 regulates contractility to control endothelial morphogenesis.</a> Mol Biol Cell. 22 (6), 795-805 (2011).</li><li>Das, 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=The+interaction+between+Shroom3+and+Rho-kinase+is+required+for+neural+tube+morphogenesis+in+mice.">The interaction between Shroom3 and Rho-kinase is required for neural tube morphogenesis in mice.</a> Biol Open. 3 (9), 850-860 (2014).</li><li>Dvorsky, R., Blumenstein, L., Vetter, I. R., Ahmadian, M. R. <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+interaction+of+ROCKI+with+the+switch+regions+of+RhoA.">Structural insights into the interaction of ROCKI with the switch regions of RhoA.</a> J Biol Chem. 279 (8), 7098-7104 (2004).</li><li>Mohan, 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=Structure+of+a+highly+conserved+domain+of+Rock1+required+for+Shroom-mediated+regulation+of+cell+morphology.">Structure of a highly conserved domain of Rock1 required for Shroom-mediated regulation of cell morphology.</a> PLoS One. 8 (12), e81075 (2013).</li><li>Mohan, 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=Structure+of+Shroom+domain+2+reveals+a+three-segmented+coiled-coil+required+for+dimerization,+Rock+binding,+and+apical+constriction.">Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction.</a> Mol Biol Cell. 23 (11), 2131-2142 (2012).</li><li>Tu, 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=Crystal+structure+of+a+coiled-coil+domain+from+human+ROCK+I.">Crystal structure of a coiled-coil domain from human ROCK I.</a> PLoS One. 6 (3), e18080 (2011).</li><li>Sievers, F., 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=Fast,+scalable+generation+of+high-quality+protein+multiple+sequence+alignments+using+Clustal+Omega.">Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.</a> Mol Syst Biol. 7, 539 (2011).</li><li>Shapiro, A. L., Vinuela, E., Maizel, J. 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+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Biophys Res Commun. 28 (5), 815-820 (1967).</li><li>Diezel, W., Kopperschlager, G., Hofmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+procedure+for+protein+staining+in+polyacrylamide+gels+with+a+new+type+of+Coomassie+Brilliant+Blue.">An improved procedure for protein staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue.</a> Anal Biochem. 48 (2), 617-620 (1972).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+limitations+of+clear-native+PAGE.">Advantages and limitations of clear-native PAGE.</a> Proteomics. 5 (17), 4338-4346 (2005).</li><li>Jungbauer, A., Hahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-exchange+chromatography.">Ion-exchange chromatography.</a> Methods Enzymol. 463, 349-371 (2009).</li><li>Yu, C. M., Mun, S., Wang, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+the+effects+of+reversible+dimerization+in+size+exclusion+chromatography.">Theoretical analysis of the effects of reversible dimerization in size exclusion chromatography.</a> J Chromatogr A. 1132 (1-2), 98-108 (2006).</li><li>Giege, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+historical+perspective+on+protein+crystallization+from+1840+to+the+present+day.">A historical perspective on protein crystallization from 1840 to the present day.</a> FEBS J. 280 (24), 6456-6497 (2013).</li><li>Lobley, A., Sadowski, M. I., Jones, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pGenTHREADER+and+pDomTHREADER:+new+methods+for+improved+protein+fold+recognition+and+superfamily+discrimination.">pGenTHREADER and pDomTHREADER: new methods for improved protein fold recognition and superfamily discrimination.</a> Bioinformatics. 25 (14), 1761-1767 (2009).</li><li>Marsden, R. L., McGuffin, L. J., Jones, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+protein+domain+assignment+from+amino+acid+sequence+using+predicted+secondary+structure.">Rapid protein domain assignment from amino acid sequence using predicted secondary structure.</a> Protein Sci. 11 (12), 2814-2824 (2002).</li><li>Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., Sternberg, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Phyre2+web+portal+for+protein+modeling,+prediction+and+analysis.">The Phyre2 web portal for protein modeling, prediction and analysis.</a> Nat Protoc. 10 (6), 845-858 (2015).</li><li>Elsliger, M. 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=The+JCSG+high-throughput+structural+biology+pipeline.">The JCSG high-throughput structural biology pipeline.</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 66 (Pt 10), 1137-1142 (2010).</li><li>Bach, 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=Escherichia+coli+maltose-binding+protein+as+a+molecular+chaperone+for+recombinant+intracellular+cytoplasmic+single-chain+antibodies.">Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies.</a> J Mol Biol. 312 (1), 79-93 (2001).</li><li>Strong, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+structural+genomics+of+complexes:+crystal+structure+of+a+PE/PPE+protein+complex+from+Mycobacterium+tuberculosis.">Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis.</a> Proc Natl Acad Sci U S A. 103 (21), 8060-8065 (2006).</li><li>Heras, B., Martin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-crystallization+treatments+for+improving+diffraction+quality+of+protein+crystals.">Post-crystallization treatments for improving diffraction quality of protein crystals.</a> Acta Crystallogr D Biol Crystallogr. 61 (Pt 9), 1173-1180 (2005).</li></ol>

Le protocole décrit ici est conçu pour aider l&#39;utilisateur à identifier les limites du domaine au sein de grandes protéines superhélice pour faciliter leur cristallisation. Le protocole repose sur une intégration globale d&#39;une série de données à partir des prédictions de calcul et d&#39;autres sources pour générer une série de limites des domaines potentiels. Ils sont suivis par une série d&#39;expériences biochimiques qui sont rapides et peu coûteux, et qui sont utilisés pour affiner ces premi?...

Détermination de la structure par cristallographie aux rayons X a apporté des contributions fondamentales à tous les domaines de la biologie moderne; fournissant une vue atomique des macromolécules qui soutiennent la vie et comment ils interagissent les uns avec les autres dans une variété de contextes; ce qui nous permet de comprendre les mécanismes qui causent les possibilités de maladies et de fournir à la conception rationnelle des médicaments pour traiter la maladie. Cristallographie a longtemps été la technique expérimentale dominante pour déterminer la structure macromoléculaire, et représente actuellement 89,3% de la base de données structurelle (www.rcsb.org). Cette technique présente de nombreux avantages, y compris le potentiel de très haute résolution, la possibilité de visualiser des macromolécules avec un large éventail de tailles, la collecte de données relativement facile, et la possibilité de visualiser comment la macromolécule interagit avec le solvant ainsi que des ligands. Malgré de nombreuses améliorations technologiques dans l&#39; expression de protéines recombinantes 1,2, purification 3, et la biologie moléculaire utilisé pour générer ces systèmes 4, le principal obstacle dans le processus cristallographique reste la capacité à produire des cristaux de qualité de diffraction. Cela a été particulièrement vrai pour les protéines qui contiennent de grands domaines coiled-coil. Il a été estimé que jusqu&#39;à 5% de tous les acides aminés se trouvent dans les bobines enroulées 5,6, ce qui en fait une caractéristique structurelle très commun 7, mais ces protéines sont souvent plus difficiles à purifier et cristalliser que les protéines globulaires 8-10 . Ceci est encore aggravé par le fait que les domaines bispiralés sont souvent trouvées dans le contexte d&#39;une protéine plus grande, par conséquent prédire correctement les limites de ces domaines est essentielle pour éviter l&#39;inclusion d&#39;une séquence non structurée ou flexible qui est souvent préjudiciable pour la cristallisation. Ici, nous présentons un cadre conceptuel combinant calcul basé sur le Web avec des analyses experimental données sur le banc, pour aider les utilisateurs de guidage à travers les étapes initiales du processus cristallographique, notamment: comment sélectionner fragment (s) de protéines pour des études structurales, et la façon de préparer et de caractériser des échantillons de protéines avant les tentatives de cristallisation. Nous concentrons notre analyse sur deux protéines contenant de grands domaines coiled-coil, Shroom (SHRM) et Rho-kinase (Rock). Ces protéines ont été choisis car ils contiennent tous les deux des domaines bispiralés et sont connues pour former un complexe biologiquement pertinente 11-16. Shroom et Rho-kinase (Rock) sont prévus pour contenir ~ 200 et 680 résidus de superhélice respectivement, de nombreuses portions qui ont été caractérisés structurellement 17-20. La méthode décrite ici fournit un flux de travail simplifié pour identifier rapidement des fragments de superhélice contenant des protéines qui se prêter à la cristallisation, cependant, les techniques décrites peuvent être facilement adaptés pour la plupart des protéines ou complexes protéiques ou modifiés pour incorporer à haut débit approChes comme disponible. Enfin, ces méthodes sont généralement peu coûteux et peuvent être effectuées par les utilisateurs à presque tous les niveaux d&#39;expérience.

<table border="0" cellpadding="0" cellspacing="0" width="925">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Rosetta</td>
			<td style="width:263px;">Emd Millipore</td>
			<td style="width:136px;">70954-3</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Star</td>
			<td style="width:263px;">ThermoFisher Scientific</td>
			<td style="width:136px;">C601003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Codon Plus</td>
			<td style="width:263px;">Agilent Technologies</td>
			<td style="width:136px;">230245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lysozyme</td>
			<td style="width:263px;">Spectrum Chemical Mfg Corp</td>
			<td style="width:136px;">L3008-5GM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ni-NTA resin</td>
			<td style="width:263px;">Life Technologies</td>
			<td style="width:136px;">25216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">SubtilisinA</td>
			<td style="width:263px;">Spectrum Chemical Mfg Corp</td>
			<td style="width:136px;">S1211-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well Cryschem Plate</td>
			<td>Hampton research</td>
			<td style="width:136px;">HR3-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INTELLI-PLATE &#160;96:</td>
			<td style="width:263px;">Art Robbins Instruments</td>
			<td style="width:136px;">102-0001-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 3350</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 8000</td>
			<td style="width:263px;">Hampton research</td>
			<td style="width:136px;">HR2-515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 400</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 4000</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">pcDNA3.1-Clover-mRuby2</td>
			<td style="width:263px;">Addgene</td>
			<td style="width:136px;">49089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Overnight Express Autoinduction System 1</td>
			<td style="width:263px;">Emd Millipore</td>
			<td>71300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysogeny Broth powder</td>
			<td style="width:263px;">ThermoFisher Scientific</td>
			<td>12795027&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

combining wet and dry lab techniques to guide the crystallization of large coiled-coil containing proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

Un diagramme illustrant le flux utilisé dans ce système est représenté sur la figure 1 et comprend trois étapes principales. analyse computationnelle de la séquence est utilisée pour formuler des hypothèses sur les limites du domaine de la protéine superhélice d&#39;intérêt. Un exemple d&#39;une analyse annotée du domaine Shrm2 SD2 est représenté sur la figure 2. Dans ce schéma, l&#39;objectif était d&#39;identifier les limites possibles...

Watch this Scientific Journal Video about Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins at JoVE.com

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

La combinaison humide et Techniques de laboratoire sec Guide pratique du Cristallisation de grandes superhélice contenant des protéines

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

Research

JoVE Journal

Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilisation, Expression, purification et cristallisation du Serotonin Human Transporter Bound to<em&gt; S</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Recherche

Biochimie

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±

Un Guide pour la Production, la cristallisation et détermination de la Structure des IKK1 humaine/α

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

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

Expression, Purification, Cristallisation et Essais enzymatiques des protéines contenant du domaine de l'hydrolase fumarylacetoacetate

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

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Utilisation d’un élément Alu contenant des minigènes pour analyser les ARN circulaires

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Sectionnement transversal du riz mature (Oryza sativa L.) Noyaux pour l’imagerie par microscopie électronique à balayage à l’aide d’embouts de pipette comme support d’immobilisation

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

Évaluation rapide de la qualité des protéines membranaires par chromatographie d’exclusion de taille fluorescente

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography (Video) | JoVE 

Criblage à haut débit pour obtenir des résultats cristallins pour la cristallographie des protéines

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

Formulation et caractérisation d’agents bioactifs contenant des nanodisques

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots (Video) | JoVE

Une approche globale pour analyser les composants cellulaires des caillots sanguins cérébraux

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...