Bu çalışma NIH hibe GM073197 ve RR025336 parçalar halinde finanse edildi.

Mezo kristalizasyon deneyde bir tipik bir anahat, Şekil 1&#39;de 1,2 gösterilmiştir . Ön kristalleşme LCP sıkı bağlamak deneyleri isteğe bağlıdır, ancak, önemli ölçüde, özellikle zor zarı proteinleri 3 durumda, ilk kristalizasyon koşulları için arama sürecini hızlandırabilir . 1. LCP içinde Protein Sulandırma <ol><li> Bir deterjan solüsyona ilgi bir membran proteini arındırmak ve konsantre ~ 10 protein / deterjan kompleksleri - 20 mg / ml, aşırı-konsantre deterjan 1,4 bakım alarak. </li><li> Transfer ~ 25 mg bir LCP ana lipid (genellikle monoolein) veya 40 1.5 ml plastik tüp ve inkübe bir lipid karışımı ° C&#39;de birkaç dakika lipid eriyene kadar. </li><li> 100 mcL gaz sıkı şırınga için bir şırınga coupler takın. </li><li> Ayarlanabilir bir hacimde pipet kullanarak erimiş lipid şırınga yükleyin. Şırınga içinde lipid hacmi kaydedin. </li><li> Bir protein çözüm-lipid oranı 2 / 3 v / protein solüsyonu ile başka bir 100 mcL şırınga Yük v. </li><li> Birlikte her iki şırınga şırınga coupler üzerinden bağlayın. </li><li> Şırınga, lipid mesophase homojen hale gelinceye kadar, ileri ve geri Kuplörün iç iğne ile lipid ve protein hareket dönüşümlü olarak pistonları itin. LCP mekanik karıştırma üzerine kendiliğinden formları ve protein LCP, çift katlı lipid sulandırılmış olur. LCP oluşumu, küçük açılı X-ışını kırınımı 1 ile çapraz polarize ile donatılmış bir mikroskop altında bakıldığında, eğer mümkünse, saydam ve jel gibi bir tutarlılık ve birefringency yokluğunda tarafından doğrulanmış, ya da olabilir. </li></ol> 2. LCP-sıkı bağlamak Ön kristalleşme Tahliller LCP-sıkı bağlamak testlerin tarama koşulları 3 çeşitli LCP sulandırılmış membran proteinlerinin difüzyon özelliklerini ölçmek için tasarlanmıştır. LCP zarı proteinleri uzun menzilli difüzyon başarılı kristalizasyon için gerekli, ancak LCP mikro büyük proteinler veya oligomerik protein agrega difüzyon kısıtlar. Mezo kristalizasyon deneyi bir başarısızlığı için ortak bir nedeni difüzyon kaybına yol açan hızlı bir protein agregasyonu. Bir protein toplama davranışı özellikle protein yapı, ev sahibi lipid ve tarama çözümü 3 bileşimi bağlı olduğu görülmüştür. <ol><li> Etiket, proteinin floresan boya ile (Cy3 veya benzeri) bir protein / boya ~ 100 / 1 oranında girmemiş boya kaldırmak ve ~ 1 mg / ml protein konsantresi. Etiket ya serbest aminler veya ücretsiz tiyoller. Ücretsiz aminler etiketleme, ağırlıklı olarak serbest N-terminalindeki etiket, 7 ve 7.5 arasında pH kullanın. Amino etiketleme lipidler co-saflaştırılmış protein 2,3 ile etiket farkında olun. </li><li> Sulandırın 1. bölümde açıklandığı gibi LCP etiketli protein). </li><li> Tahlil plakalar yerine kristalizasyon ekranlarında 2 LCP-sıkı bağlamak tarama çözümleri kullanarak) 3. bölümünde açıklandığı gibi ayarlayın. </li><li> 20 plakalar inkübe ° C denge durumuna ulaşmak için en az 12 saat süreyle karanlıkta. </li><li> 10x objektif kullanarak ilk LCP sıkı bağlamak istasyonu ve odak yerleştirin plakalar. </li><li> Ilk ön-ağartılmış durumunu yakalamak için 5 floresan görüntüler elde. </li><li> Lazer tetikler. Ağartılmış spot ortasında etiketli protein% 70 - çamaşır suyu ~ 30 bakliyat lazer gücü ve sayısı ayarlanabilir olmalıdır. </li><li> Lazer tetikleme hemen sonra, mümkün olan en hızlı hızı ~ 200 resimler hızlı bir post-beyazlatma dizisi kaydetmeye başlamak. </li><li> Protein difüzyon hızı bağlı olarak 1-20 sn gibi görüntüler arasında gecikme seçerek ~ 50 resimler, yavaş bir post-çamaşır suyu dizisi kaydı ile izleyin. </li><li> Tüm çerçeveler içinde çamaşır suyu nokta yoğunluğu içinde entegre ve lazer ağartılmış alanının dışında bir referans nokta ortalama yoğunluğu, ağartılmış nokta içinde yoğunluğu bölerek satın alma sırasında yoğunluk dalgalanmaları ağartma ve ışık için doğru. </li><li> 1&#39;e eşit önceden ağartılmış yoğunluğu ve ilk ağartılmış yoğunluğu 0&#39;a eşit yapmak için düzeltilmiş yoğunluk Normale. </li><li> Aşağıdaki denklem 5 kullanılarak normalize yoğunluğu vs zaman, F (t) eğrisi Fit: F (t) = M x exp (-2T / t) x (I 0 (2T / t) + 1 (2T / t)), (Eq.1) M difüzyon moleküllerin cep fraksiyonu, T karakteristik difüzyon süresi mi, 0 kaydedilen her karenin gerçek zamanlı ve ben 1 0. ve 1. sırası Modifiye Bessel fonksiyonları. </li><li> Difüzyon katsayısı, D gibi hesaplayın: D = R 2 / 4T (Eq.2) R ağartılmış spot yarıçapı. </li><li> T Taşıo 2.13) yanında iyi ve adımları tekrarlayın 2.5). </li><li> Farklı tarama koşulları için elde edilen mobil kesirler ve difüzyon katsayıları karşılaştırın. Tasarım yeni kristalizasyon protein difüzyon ve protein difüzyon görülmedi hangi hariç koşulları kolaylaştırılmış bileşenleri dayalı ekranlar. Protein ekranlı durumlardan herhangi diffüz olmadıysa, tarama alanı genişletilmesi veya yeni bir protein inşa çalışıyor düşünün. </li></ol> 3. LCP Kristalizasyon Denemeler Ayarlama <ol><li> LCP içinde sulandırın protein gibi) 1 bölümünde açıklanan. </li><li> Protein yüklü LCP tekrarlayan bir şırınga dağıtıcı bağlı bir gaz sızdırmaz 10 mcL şırınga içine aktarın. </li><li> 10 mcL şırınga, kısa bir çıkarılabilir iğne (gösterge 26, 10 mm uzunluk) takın. </li><li> 2x2 bir kare oluşturan bitişik dört kuyu yüzeyinde LCP 200 nL bolus koyun. </li><li> 1 mcL ilgili kristalizasyon ekran çözümü ile her LCP bolus Overlay. </li><li> Cap dört bir 18 mm kare cam lamel yüklü kuyu. Kuyu mühür lamel üzerine nazik bir basınç uygulayın. </li><li> 4 kuyu sonraki bütün plaka dolana kadar) 3.4) -3,6 adımları tekrarlayın. </li><li> Kristal oluşumu ve büyümesi için periyodik olarak kontrol, tabak sabit bir sıcaklıkta inkübe edin. </li></ol> 4. LCP gelen Kristaller Hasat <ol><li> Doğrusal bir döner polarize ve analiz ile donatılmış bir stereo mikroskop altında değişken zoom ile protein kristalleri ile bir tabak koyun. </li><li> Bütün iyi görüş alanı içinde yer almakta olup, böylece düşük güç zoom kullanırken ilgi iyi odaklanın. </li><li> Keskin bir köşe, seramik kılcal kesme taş kullanılarak iyi sınırları içinde bir kare dört vuruş lamel cam Puan. </li><li> Lamel cam kalınlığı boyunca çizikler yaymak için güçlü keskin nokta cımbız ile gol çevresinde basın. </li><li> Punch iki küçük delik attı kare ters köşe. </li><li> Yağışlı çözüm birkaç mcL sonraki adımları sırasında dehidrasyon azaltmak için bir delik aracılığıyla enjekte edilir. </li><li> Açılı keskin bir iğne probu kullanarak cut-out kare serbest bırakmak için bir ya da iki tarafı boyunca cam kırmak. </li><li> Kübik faz bolus cam kare izlenme dikkatlice yukarı doğru kaldırın. Bolus lamel sıkışmış ise, o kuyunun altındaki kare yer ve cam üzerine çevirin. </li><li> Yağışlı bir çözüm ekstra birkaç mcL ile desteklenmiş bir cryo-Koruyucu, gerekirse, kuyu maruz küp faz bolus üstüne. </li><li> Mikroskop büyütme artırın ve bir kristal odaklanmak. </li><li> Hasat döngüsü görmek için yeterli ışık tutarken, birefringent kristal ve arka plan arasındaki kontrastı arttırmak için polarize ve analizör arasındaki açıyı ayarlayın. </li><li> MiTeGen MicroMount kristal boyutu ile eşleşen bir çapa sahip bir seçin ve sonra MicroMount içine scooping LCP doğrudan kristal hasat. </li><li> Flash sıvı azot hasat kristal MicroMount dondurma, ve, X-ışını veri toplama 6 için sinkrotron kaynağı beamline gemi . </li></ol> 5. Temsilcisi Sonuçlar: Bir mühendislik insan beta-2 adrenerjik G protein kenetli reseptör (β 2 AR-T4L) bakülovirüs sf9 böcek hücreleri enfekte dodecylmaltoside (DDM) / 7 carazolol kısmi bir ters agonisti bağlı kolesteril hemisuccinate (CHS) deterjan solüsyonu saflaştırılmış ifade edildi. Protein Cy3 NHS ester ile etiketlenir ve LCP-sıkı bağlamak kristalleşme öncesi testleri (Şekil 2) kullanılmıştır. LCP-sıkı bağlamak testlerinin sonuçları seçilen çeşitli koşullar dayalı Kaba ızgara ekranları ilk kristal gibi vurur (Şekil 3). Yağışlı koşullarının daha fazla optimizasyon kırınım kalite kristalleri (Şekil 4) vermiştir. <img src="/files/ftp_upload/2501/2501fig1.jpg" alt="Şekil 1" /> Şekil 1 tipik bir LCP kristalizasyon deneyi Akış şeması. Gri kutular Adımlar mevcut protokoller açıklanan değildir. <img src="/files/ftp_upload/2501/2501fig2.jpg" alt="Şekil 2" /> Şekil 2 monoolein tabanlı LCP β 2 AR-T4L/carazolol LCP-sıkı bağlamak tahlil. A) LCP-sıkı bağlamak testinin sonuçları, 96 plaka her örnek sırayla ağartılmış ve 30 dakika inkübasyondan sonra floresan kurtarma ölçüldüğü, yüksek verimli otomatik modda. Her bir numune mobil bölümünü temsil edilen floresan kurtarma, 96 numuneler için çizilir. Tarama çözümleri iki farklı konsantrasyonda az 48 farklı tuzları ile birlikte 0,1 M Tris pH: 8,% 30 v / v PEG 400 içerir. B) birkaç temsilcisi Floresan kurtarma profilleriresentative koşulları. Düz çizgi eğrileri Denklem tarafından uyuyor temsil eder. 1. cep kesirler ve difüzyon katsayıları Denklem kullanılarak belirlenir. 1 ve 2. ,% 10 daha az Na klorür içeren örnek hızlı iyileşme co-saflaştırılmış protein ile floresan etiketli lipidler nedeniyle. <img src="/files/ftp_upload/2501/2501fig3.jpg" alt="Şekil 3" /> Şekil 3 β 2 AR-T4L/carazolol ilk kristal hit LCP-sıkı bağlamak tarafından belirlenen en umut verici koşulları etrafında, sülfat Na içeren bir kaba ızgara tarama ile elde edilen (panel) ve Na biçimleri (panel). Protein Cy3 NHS ester ile etiketlenir ve 605 nm eksitasyon ve 543 nm dalga boyunda emisyon kullanarak floresan görüntüler alınır. <img src="/files/ftp_upload/2501/2501fig4.jpg" alt="Şekil 4" /> Şekil 4. β 2 AR-T4L/carazolol Optimize edilmiş kristalleri. (Paneller A ve B), Na sülfat varlığı yetiştirilen kristalleri ve K biçimleri (panel C ve D) görüntüleri aydınlık modu (paneller A ve C) ve çapraz-polarize (paneller B ve D).

<ol>
	<li>Caffrey, M., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallizing+membrane+proteins+using+lipidic+mesophases.">Crystallizing membrane proteins using lipidic mesophases.</a> Nat. Protoc. 4, 706-731 (2009).</li><li>Cherezov, V., Abola, E., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+structure+determination+of+GPCRs,+a+membrane+protein+family+with+high+potential+as+pharmaceutical+targets.">Recent progress in the structure determination of GPCRs, a membrane protein family with high potential as pharmaceutical targets.</a> Methods Mol. Biol. 654, 141-170 (2010).</li><li>Cherezov, V., Liu, J., Hanson, M. A., Griffith, M. T., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LCP-FRAP+assay+for+pre-screening+membrane+proteins+for+in+meso+crystallization.">LCP-FRAP assay for pre-screening membrane proteins for in meso crystallization.</a> J. Cryst. Growth Design. 8, 4307-4315 (2008).</li><li>Misquitta, Y., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergents+destabilize+the+cubic+phase+of+monoolein:+implications+for+membrane+protein+crystallization.">Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization.</a> Biophys. J. 79, 394-405 (2003).</li><li>Soumpasis, D. M. <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+fluorescence+photobleaching+recovery+experiments.">Theoretical analysis of fluorescence photobleaching recovery experiments.</a> Biophys. J. 41, 95-97 (1983).</li><li>Cherezov, V., Hanson, M. A., Griffith, M. T., Hilgart, M. C., Sanishvili, R., Nagarajan, V., Stepanov, S., Fischetti, R. F., Kuhn, P., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rastering+strategy+for+screening+and+centering+of+microcrystal+samples+of+human+membrane+proteins+with+a+sub-10+micrometer+size+X-ray+synchrotron+beam.">Rastering strategy for screening and centering of microcrystal samples of human membrane proteins with a sub-10 micrometer size X-ray synchrotron beam.</a> J. R. Soc. Interface. 6, S587-S597 (2009).</li><li>Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H. J., Kuhn, P., Weis, W. I., Kobilka, B. K., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+crystal+structure+of+an+engineered+human+beta2-adrenergic+G+protein-coupled+receptor.">High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.</a> Science. 318, 1258-1265 (2007).</li><li>Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y. F., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robotic+system+for+crystallizing+membrane+and+soluble+proteins+in+lipidic+mesophases.">A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases.</a> Acta Crystallogr. D. 60, 1795-1807 (2004).</li><li>Kissick, D. J., Gualtieri, E. J., Simpson, G. J., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+optical+imaging+of+integral+membrane+protein+crystals+in+lipidic+mesophases.">Nonlinear optical imaging of integral membrane protein crystals in lipidic mesophases.</a> Anal. Chem. 82, 491-497 (2010).</li><li>Cheng, A., Hummel, B., Qiu, H., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+mechanical+mixer+for+small+viscous+lipid-containing+samples.">A simple mechanical mixer for small viscous lipid-containing samples.</a> Chem. Phys. Lipids. 95, 11-21 (1998).</li><li>Cherezov, V., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+inexpensive+nanoliter-volume+dispenser+for+highly+viscous+materials+used+in+membrane+protein+crystallization.">A simple and inexpensive nanoliter-volume dispenser for highly viscous materials used in membrane protein crystallization.</a> J. Appl. Cryst. 38, 398-400 (2005).</li><li>Cherezov, V., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-volume+plates+with+excellent+optical+properties+for+fast,+inexpensive+crystallization+screening+of+membrane+proteins.">Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins.</a> J. Appl. Cryst. 36, 1372-1377 (2003).</li><li>Cherezov, V., Fersi, H., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+screens:+compatibility+with+the+lipidic+cubic+phase+for+in+meso+crystallization+of+membrane+proteins.">Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins.</a> Biophys J. 81, 225-242 (2001).</li></ol>

Protokolleri mezo kristalizasyon deneyler yapılması dahil temel adımlar için bir temel görsel rehberlik sağlar. Derinliği olası tuzaklara vurgulayarak, bu protokoller ile ilgili detaylar daha fazlası, bazı eksiklikler veya başka alternatif güzergahlar 1,2 mevcuttur. İsteğe bağlı LCP-sıkı bağlamak testlerin en çok gelecek vaat eden bir protein yapısı, LCP ana lipid ve lipid katkı yanı sıra mümkün precipitants ve tampon koşulları 3 sınırlandırmayı seçmek için önceki aşamalarında yardımcı olabilir. Ilk kristalizasyon isabet sonra, daha kaliteli kristaller elde etmek için optimize edilmelidir. Mezo kristalizasyon koşullarının optimizasyonu LCP 1 bileşimi ile ilişkili ilave parametrelerin ilavesi ile çözünür proteinler için iyi duruma getirmek için temelde benzer. Lipidik mesophase yetiştirilen Membran protein kristalleri genellikle deterjan solüsyonu elde edilen kristaller daha küçüktür, ama daha modern bir sinkrotron kaynakları 6 mikro fokus kullanarak güçlü yararlanarak, böylece mevcut beamlines emretti. Kristalizasyon veya tahlil plakaları kurma LCP-sıkı bağlamak deneyleri yürüten ve kristaller tespit mezo kristalizasyon, ilgili prosedürlerin çoğu sağlayan, 1,2,8,9 yarı veya tam otomatik olan koşulları daha geniş bir yelpazede tarama protein ve lipid tüketen küçük miktarda. Öte yandan, LCP ve kristal hasat protein reconstitutions manuel ve sıkıcı işlemleri kalır ve böylece iyileştirilmesi için bir ihtiyaç var.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>100 &mu;L gas-tight syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7656-01</td>
<td>2 syringes are required</td>
</tr>
<tr>
<td>10 &mu;L gas-tight syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7653-01</td>
<td>&nbsp;</td>
<tr>
<td>Syringe coupler</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7770-02 30902</td>
<td>The coupler can be made using available parts as described in refs. 10</td>
</tr>
<tr>
<td>Repetitive syringe dispenser</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>83700</td>
<td>The repetitive syringe dispenser can be modified to reduce dispensing volume by ~3 times11</td>
</tr>
<tr>
<td>Short (0.375&#x201D;) flat-tipped removable needle (point style 3, gauge 26)</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7804-03</td>
<td>&nbsp;</td>
<tr>
<td>96-well glass sandwich plate</td>
<td>&nbsp;</td>
<td>Marienfeld</td>
<td>08 900 03</td>
<td>For manual operations it is more convenient to assemble the glass sandwich plate using a standard microscope glass slides and a double sticky tape with punched holes1,2,12.</td>
</tr>
<tr>
<td>Glass cover slip</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>63787-01</td>
<td>&nbsp;</td>
<tr>
<td>monoolein</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7765</td>
<td>&nbsp;</td>
<tr>
<td>Crystallization screens</td>
<td>&nbsp;</td>
<td>Hampton Research, Molecular Dimensions, Emerald Biosystems, Jena Bioscience</td>
<td>&nbsp;</td>
<td>Most of available commercial screens can be used for initial screening. Conditions that consistently disrupt LCP can be diluted 2x for better compatibility13.</td>
</tr>
<tr>
<td>Capillary cutting stone</td>
<td>&nbsp;</td>
<td>Hampton Research</td>
<td>HR4-334</td>
<td>&nbsp;</td>
<tr>
<td>Fine point tweezers</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>510</td>
<td>&nbsp;</td>
<tr>
<td>Angled sharp probe</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13650</td>
<td>&nbsp;</td>
<tr>
<td>MicroMounts</td>
<td>&nbsp;</td>
<td>MiTeGen</td>
<td>M1-Lxx-xx</td>
<td>Select MicroMount diameter to match the crystal size</td>
</tr>		</tbody>
		</table>
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crystallization of membrane proteins in lipidic mesophases

Zar proteinleri, sinyal iletimi, ulaşım ve enerji dönüşümleri, gibi çok sayıda arıza ve hastalıkların karıştığı ile ilgili canlı hücreler kritik işlevleri yerine. Ancak, membran proteinlerinin yapısal ve işlevsel bir anlayış güçlü kırınım kaliteli kristaller çözünürleştirme ve üretim ile ilgili zorluklar nedeniyle, özellikle, kendi çözünür ortakları geride kalıyor. Lipidik mesophases Kristalizasyon (mezo ya da LCP kristalizasyon olarak da bilinir), mikrobiyal rhodopsins fotosentetik proteinler, dış membran beta varil ve G-protein-bağlı reseptörler yüksek çözünürlüklü yapılar elde etmek için başarıyla uygulanmıştır gelecek vaat eden bir tekniktir . Mezo olarak kristalizasyon anadili gibi bir membran çevre yararlanır ve genellikle düşük solvent içeriği ve deterjan geleneksel kristalizasyon ile karşılaştırıldığında daha iyi sipariş ile kristaller üretir. Bu yöntem zor değildir, ancak, viskoz mesophase malzeme taşıma lipid faz davranışları ve pratik bir anlayış gerektirir. Burada LCP yapma ve bir membran proteini kullanarak LCP, bir tahlil veya kristalizasyon plaka LCP nanoliter bölümlerini dağıtım, kristalleşme öncesi testlerin yürütülmesi ve kristaller hasat bir şırınga mikser, çift katlı lipid yeniden oluşturmak basit ve verimli bir şekilde göstermektedir LCP matris. Ancak, herhangi bir kristalizasyon deneyi, geniş tarama ve optimizasyon gibi gerekli ve başarılı bir sonuç mutlaka garanti değildir; Bu protokoller mezo kristalizasyon çalışmalarda yaklaşan için temel bir kılavuz sağlar.

Watch this Scientific Journal Video about Crystallization of Membrane Proteins in Lipidic Mesophases at JoVE.com

Crystallization of Membrane Proteins in Lipidic Mesophases

Protokolleri LCP kristalizasyon denemeler ve hasat kristalleri kurma, LCP-sıkı bağlamak kristalleşme öncesi testlerin başlangıç ​​koşulları bulma lipidik kübik bir fazlı (LCP) protein, sulandırıldıktan itibaren bir membran proteini kırınım kaliteli kristaller elde etmek için gerekli adımları açıklar .

Lipidik Mesophases Membran Proteinleri Kristallendirilmesi

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

crystallization-of-membrane-proteins-in-lipidic-mesophases

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JoVE Journal

Biology

31.2K Görüntüleme.  The Scripps Research Institute. Protokolleri LCP kristalizasyon denemeler ve hasat kristalleri kurma, LCP-sıkı bağlamak kristalleşme öncesi testlerin başlangıç ​​koşulları bulma lipidik kübik bir fazlı (LCP) protein, sulandırıldıktan itibaren bir membran proteini kırınım kaliteli kristaller elde etmek için gerekli adımları açıklar .

Video: Crystallization of Membrane Proteins in Lipidic Mesophases

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Lipidik Mesophases kullanarak Yapı Tayini için Membran Proteinleri Kristalize

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Biyoloji

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Elektron Kristalografi Yapısal Biyoloji Araştırmaları Küçük Membran Proteinleri İki boyutlu Kristalizasyon Denemeler değerlendirilmesi

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Membran Proteinleri Konformasyonel Dynamics incelenmesi In situ Site Floresan Etiketleme

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Yüksek verim Kristalizasyon lipidik Bicelle Yöntemiyle Membran Proteinleri

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidik Mesophases Membran Proteinleri Yüksek throughput Kristalizasyon için bir robot kullanımı

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Makromoleküler Kristalografi tarafından Yapı Tayini için lipidik Mesophases Yetiştirilen Membran Proteinlerinin Hasat ve Cryo-soğutma Kristaller

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Floresan Proteinler ifade Canlı Farelerde Görüntüleme hücrealtı Yapıların Intravital Mikroskopi

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

İzole Mitokondri Akış Sitometrisi ile Dış Zar Proteinleri imüno

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Membran proteinlerinin Yeşil floresan proteini temelli Ekspresyon elemesinde<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Şekilde, mikro Tekniği Sıçan mezenter mikrodamar geçirgenliği Yönetmelik Araştırma

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Methods Article

Lipidik Mesophases Membran Proteinleri Kristallendirilmesi