Bu çalışma bir Burroughs Wellcome YM Araştırmacı Ödülü ve MD, Yale Üniversitesi&#39;nde Brown-Coxe Doktora Sonrası Araştırma Bursu ile desteklenmiştir.

Malzemeler: <ul><li> Protein örnek - Lizozim (50 mg / ml) </li><li> Bırak 24-iyi tepsi Asma </li><li> Oturma açılan 24 kuyu tepsisi </li><li> Microbatch kristalizasyon 96 tepsi </li><li> Kristalizasyon çözümler (ya ticari ya da ev yapımı) </li><li> Silikon yağ </li><li> Luer-lock olmadan 5 ml şırınga </li><li> Silikonlu kapak kızakları </li><li> Optik sızdırmazlık bantı </li><li> Parafin </li><li> 0,1-2 mcL mikropipet düşük tutma ipuçları </li><li> Cımbız </li><li> Profesyonel mendil </li></ul> 1. Asma / Bırak Prosedürü oturan: <ol><li> Bir başlangıç ​​ekranı için bir deneme koşulları seyrek matris eksik faktöriyel yöntemi istismar piyasada bulunan ekranlar kullanabilirsiniz. Seyrek matris eksik faktöriyel yöntemi önyargılı ve makromoleküller için bilinen kristalizasyon koşulları (Carter ve Carter, 1979; Cudney ve ark, 1994; Jancarik ve Kim, 1991; Jancarik ve ark, 1991) seçilir. Kristalleşme durumu zaten bilinen ise, orijinal hit etrafında Yağışlı konsantrasyon ve pH değişen dar bir ekran monte edilebilir. En sık başarılı Yağışlı polietilen glikol (PEG), amonyum sülfat. Birlikte, kristalizasyon (Gilliland ve ark, 1994; Kimber ve ark, 2003; Page ve ark, 2003) için kullanılan tüm kaydedilen makromoleküler precipitants yaklaşık% 60 için bu iki precipitants hesap. </li><li> Kristalizasyon çözümler hazırlayın. Genellikle bu koşulları presipite edici bir ajan, bir tür tuz ve pH ayarlamak için bir tampon birleştirilir. Bazı durumlarda katkı için özel ilgi protein eklenebilir. Tüm stok solüsyonu 0.22 mikron filtre kullanılarak filtre edilmelidir. Bazı stok solüsyonu (% 50 PEG, gliserol, PEG-MME, vb) çok viskoz olabilir. <ul><li> 1,5 mL 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 0.1 M NaOAc pH 4.0, 4.3, 4.6 ve 4.9 M NaCl (toplam 24 çözümleri) her hazırlayın </li></ul></li><li> Protein örnek çözülme ve buz üzerinde tutmak. Protein hisse senetleri, faiz protein (eğer böyleyse, mağaza, 4 ° C) donma / çözülme duyarlı sürece -80 ° C&#39;de muhafaza edilmelidir. Tipik protein konsantrasyonları yüksek konsantrasyonlarda genellikle daha iyi sonuçlar veren, 5-50 mg / ml aralığında. </li><li> Bazı proteinler kısmi bir çözülme adımdan sonra agrega ve çökelti eğilimindedir - 15min/18, 000xg / 4 ° C örnek Spin. Bu rakamlar, bu nedenle ayrıntılı bir dönüş örnek sadece çözünür moleküller sağlayacak protein molekülleri, geri kalanı amorf yağış teşvik edebilir. Tepsi kadar buz üzerinde protein tutun. </li><li> UV spektrofotometre ile 280 nm dalga boyunda absorbans gelen protein konsantrasyonu belirleyin. Protein konsantrasyonu absorbans okuma ve protein yok katsayısı hesaplanabilir. : Yok katsayısı EXPASY web sitesinde ProtParam aracı ile hesaplanır. <a href="http://ca.expasy.org/tools/protparam.html">http://ca.expasy.org/tools/protparam.html</a> </li><li> 24-iyi asılı / damla tepsi tepsi kurulum şeması (Şekil 2B) göre Yağışlı çözüm 500 mcL oturan kuyu doldurun. Kuyunun kenarında bir silikon gres halka oluşturun. Halka iyi mühürlü olarak hava basıncı oluşumunu önlemek için küçük bir boşluk olmalıdır. Damla oturan, iyi (Şekil 2A) bandı ile mühürlenir beri gres gerek yoktur. </li><li> Bir kapak slayt atın ve yoğun hava spreyi ya da silin profesyonel bir temiz kaçınarak toz kristalizasyon damla yorumlama kolaylığı ve kontaminasyonların ortadan kaldıracaktır. Damla oturma için, kapağını kaydırarak kullanılmaz. Bunun yerine, iyi bir protein ve yağışlı karışık olduğu bir raf bulunur. </li><li> Kapak slayt örnek protein ve rezervuar çözümü eşit miktarda koyun. Farklı miktarda protein ve yağışlı optimizasyon sürecinin parçası olarak denenebilir. Seyreltik protein örneklerinin için arzu edilebilir, ve kristal çekirdeklenme veya büyüme yavaş dengeleme sonra açılan, protein net bir konsantrasyon Yağışlı sonuçlar daha fazla protein kullanılması. Açılan çözümü diğer herhangi bir kalıcı madde de aynı faktör tarafından konsantre olacaktır. Kapak slayt üzerine protein ve rezervuar çözümleri pipetleme, kabarcık oluşumunu önlemek için son derece dikkatli olun. Bu genellikle hava pipetin üflenir oluşur. <ul><li> 0.02 M NaOAc pH 4.9 50 mg / ml lizozim Yük 2 mcL kapak slayt merkezi ve rezervuar çözüm 2 mcL ekleyin: damla Hanging. </li><li> Damla Oturma: Yük 2 raf merkezine 50 mg / ml lizozim çözüm mcL ve rezervuar çözüm 2 mcL ekleyin. </li></ul></li><li> Yavaşça kapağını kaydırarak çevirin ve kuyunun üstüne yağ halkası üzerine uzandı. Pr yağ içinde hafifçe hava yoluyla iyi çentik kaçabilir bastırınolay hava basıncı birikimi ve iyi tutmak iyi mühürlenir. Hava basıncı oluşumunu iyi, iyi mühür ödün, kapağı kaydırın artırabilirsiniz. Oturma bırak yöntemi, optik saydam bant kuyuları kapatmak için kullanılmaktadır. Bu nedenle, bu yöntem sızdırmazlık, her satır / sütun yer alacak. </li><li> Tepsi tamamlanana kadar bir sonraki iyi geçin. </li><li> Kurulum üzerine tepsisini kontrol edin, toz parçacıkları ve diğer kirlilikler ortadan kaldırarak protein kristalleri yanlış pozitif kimlik azaltabilir. </li><li> Denemenizi belge için bir puanlama tablosu kullanın. </li><li> Genellikle 4 ° C ve oda sıcaklığı arasında, istenen inkübasyon sıcaklığında tepsiye yerleştirin. 20 ° C, en sık başarılı. Tepsi hafifçe ele almak ve herhangi bir sallayarak önlemek için özen gösterin. Inkübatör kapı açılış ve kapanış ederken dikkatli olun. Transfer veya inkübasyon sırasında titreşim veya sıcaklık değişiklikleri, kristal büyümesini önlemek veya kristal kalitesini olumsuz etkileyebilir. Bir şok emici malzeme (örneğin ambalaj köpük) kristal tepsileri altında dolgu olarak kullanılabilir. </li><li> Kristalleri için tepsi ertesi gün kontrol edin ve sonra her birkaç gün, her zaman dikkatli tepsileri taşıma. Bir tepsi özel puanlama sayfasında herhangi bir bulgu ve bırak morfolojileri Belge. Kristalleri bazen çok daha erken (hemen) veya sonra (birkaç ay kadar!) Görünmekle beraber Kristaller genellikle görünür, 2-5 gün sürebilir. Kristalizasyon koşulları tespit edilmiştir ve kristallerin büyüme oranı bilinmektedir sonra kristaller ortaya çıktı ve son boyutu en az yarısının büyüdü kadar, tepsiler rahatsız bırakmak en iyisidir. Tepsileri rahatsız bırakmak, dolayısıyla daha az sayıda daha büyük kristaller üreten, kristal çekirdeklenme olayların sayısını azaltabilir. </li></ol> 2. Prosedür Microbatch: <ol><li> Protein örnek ve yağışlı hazırlık yukarıda açıklandığı gibi. </li><li> Hava püskürtme yeni bir microbatch tepsisi, toz ve diğer büyük parçacıklar önlemek için. </li><li> Microbatch tepsi, 3 mm yüksekliğinde (kuyuları kapsayacak şekilde yeterli) parafin yağı doldurun. Petrol erişimi kaldırın. </li><li> Önceden doldurulmuş, yağ, protein solüsyonu (50 mg / ml lizozim) 1 mcL Yük iyi alt (Şekil 2A) doğrudan çözüm pipetle emin olun. </li><li> Açılan protein damlacık ile, iyi ve sigortalar alt lavabolar emin olun iyi tepsi kurulum şeması (Şekil 2B) göre 1 mcL Yağışlı çözüm yükleyin. </li><li> Tepsi tamamlanana kadar yanında iyi gider. </li><li> Asılı / bırak işlemi oturan 11-14 adımları. </li></ol> 3. Temsilcisi Sonuçlar: Kristalizasyon, genellikle X-ışını kristalografisi darboğaz olarak adlandırılır. Presipite koşullar seyrek matris eksik faktöriyel ekran genellikle büyük tek kristalleri arasında, protein agregasyonu ve yağış, birçok farklı türde üretir. Protein veya Yağışlı konsantrasyonları çok yüksek bir hiç farklı şekil ve boyut (amorf yağış) ile kahverengi madde görebilirsiniz. Çözüm doymuş olduğunda, açılan genellikle yağış her türlü açık ve tamamen yoksun olacaktır. Şekil 3 yağış olayları ve kristaller (Dessau ve ark, 2006) için çeşitli örnekler gösterir. Daha detaylı yorumlanması ile ilgili daha fazla yağış fenomen <a href="http://xray.bmc.uu.se/terese/tutorials.html">http://xray.bmc.uu.se/terese/tutorials.html</a> adresinde bulunabilir . <img src="/files/ftp_upload/2285/2285fig1.jpg" alt="Şekil 1" /> Şekil 1. Protein kristalizasyon ilkesi. Protein kristalizasyon ilkesi. Buharı difüzyon deneyi (A), yağışlı ve protein eşit hacimlerde açılan bulunmaktadır. Su dışına dağılacaktır ve damla ve rezervuar çözüm arasındaki denge sağlanana kadar yağışlı ve protein konsantrasyonu iki katına çıkarılacaktır. Toplu kristalizasyon (B) Yağışlı ve deney sırasında protein konsantrasyonu değişmez. A Noktası - Protein kristalleri, B noktası oluşmuş olabilir doymuş kalır - Protein çekirdeklenme meydana gelen, kristaller oluşmaya başladı çözüm protein konsantrasyonu ve doygunluk düşer. Nokta C - Protein çökeltileri, ama kristalleri hala büyümek. (C) kullanarak Diyaliz kristalizasyon kapiller jel-takılı. Kristaller tuz jel fiş boyunca (ve / veya Yağışlı yayılır protein örnek içine) protein örnek yayılır gibi görünür. <img src="/files/ftp_upload/2285/2285fig2.jpg" alt="Şekil 2" /> Şekil 2. Anahat tipik bir protein kristalizasyon deneyler. (A), damla buharı difüzyon asılı damla buharı difüzyon oturan ve microbatch protein kristalizasyon Prosedürler. Her iki durumda da, konsantre protein örnek bir küçük hacimli precipitan eşit veya daha küçük hacmi ile karıştırılır.t çözüm ve denge sağlaması için izin. (B), 24-iyi bir tepsi tavuk lizozim kristalizasyon için değişik konsantrasyonlarda sodyum klorür (Yağışlı) ve çeşitli pH tampon olarak 0.1 M sodyum asetat (4.0 - 4.9) ile ayarlanır. <img src="/files/ftp_upload/2285/2285fig3.jpg" alt="Şekil 3" /> Şekil 3. Tipik bir protein kristalizasyon deney sonuçları. (A) Amorf yağış. Protein veya yağışlı (veya her ikisi), yüksek konsantrasyonda olduğu zaman. (B) faz ayrılması. Protein veya deterjan yüksek konsantrasyonda bazı precipitants ile karıştırılarak farklı bir faz için ayrı olabilir. (C) AtCSN7 Rod şekilli protein kristalleri polietilen glikol 8000 ve elde edilen magnezyum asetat (Dessau ve ark, 2006). (D) Lizozim kristallerinin 1.0 M NaCl ve sodyum asetat pH 4.9 aldı. (E) doymuş damla altında genellikle açık kalır. Moşe Dessau Fotoğraf.

<ol>
	<li>Bergfors, T. 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> Protein crystallization : techniques, strategies, and tips : a laboratory manual. , (1999).</li><li>Carter, C. W., Carter, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystallization+using+incomplete+factorial+experiments.">Protein crystallization using incomplete factorial experiments.</a> J Biol Chem. 254, 12219-12223 (1979).</li><li>Cudney, R., Patel, S., Weisgraber, K., Newhouse, Y., McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+optimization+strategies+for+macromolecular+crystal+growth.">Screening and optimization strategies for macromolecular crystal growth.</a> Acta Crystallogr D Biol Crystallogr. 50, 414-423 (1994).</li><li>Dessau, M., Chamovitz, D. A., Hirsch, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+purification+and+crystallization+of+a+PCI+domain+from+the+COP9+signalosome+subunit+7+(CSN7).">Expression, purification and crystallization of a PCI domain from the COP9 signalosome subunit 7 (CSN7).</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 62, 1138-1140 (2006).</li><li>Gilliland, G. L., Tung, M., Blakeslee, D. M., Ladner, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Macromolecule+Crystallization+Database,+Version+3.0:+new+features,+data+and+the+NASA+archive+for+protein+crystal+growth+data.">Biological Macromolecule Crystallization Database, Version 3.0: new features, data and the NASA archive for protein crystal growth data.</a> Acta Crystallogr D Biol Crystallogr. 50, 408-413 (1994).</li><li>Jancarik, J., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparse+matrix+sampling:+a+screening+method+for+crystallization+of+proteins.">Sparse matrix sampling: a screening method for crystallization of proteins.</a> J Appl Cryst. 23, 409-411 (1991).</li><li>Jancarik, J., Scott, W. G., Milligan, D. L., Koshland, D. E., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+study+of+the+ligand-binding+domain+of+the+bacterial+chemotaxis-mediating+aspartate+receptor+of+Salmonella+typhimurium.">Crystallization and preliminary X-ray diffraction study of the ligand-binding domain of the bacterial chemotaxis-mediating aspartate receptor of Salmonella typhimurium.</a> J Mol Biol. 221, 31-34 (1991).</li><li>Kimber, M. S., Vallee, F., Houston, S., Necakov, A., Skarina, T., Evdokimova, E., Beasley, S., Christendat, D., Savchenko, A., Arrowsmith, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+mining+crystallization+databases:+knowledge-based+approaches+to+optimize+protein+crystal+screens.">Data mining crystallization databases: knowledge-based approaches to optimize protein crystal screens.</a> Proteins. 51, 562-568 (2003).</li><li>Lorber, B., Sauter, C., Theobald-Dietrich, A., Moreno, A., Schellenberger, P., Robert, M. C., Capelle, B., Sanglier, S., Potier, N., 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=Crystal+growth+of+proteins,+nucleic+acids,+and+viruses+in+gels.">Crystal growth of proteins, nucleic acids, and viruses in gels.</a> Prog Biophys Mol Biol. 101, 13-25 (2009).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Crystallization of biological macromolecules. , (1999).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+protein+crystallization.">Introduction to protein crystallization.</a> Methods. 34, 254-265 (2004).</li><li>Page, R., Grzechnik, S. K., Canaves, J. M., Spraggon, G., Kreusch, A., Kuhn, P., Stevens, R. C., Lesley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+crystallization+strategy+for+structural+genomics:+an+optimized+two-tiered+crystallization+screen+against+the+Thermotoga+maritima+proteome.">Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome.</a> Acta Crystallogr D Biol Crystallogr. 59, 1028-1037 (2003).</li></ol>

Bu yazıda, protein kristalizasyon için genel geçerli protokolleri anlatmak ve göstermek. Çok aşamalı bir prosedür bu yana birkaç düşünceler farkında olmak bir ihtiyacı vardır. Buharlaşma nedeniyle açılan kuruma (0.5-2 mcL), çok küçük hacimleri ile çalışırken, büyük bir endişe. Bu nedenle, iyi-kontrollü bir ortamda çalışmak (düşük hava akımı, yüksek nem ve sıkı sıcaklık kontrolü ile) ve açık havaya açılan maruz kalma en aza indiren bir tekniktir benimsemeye tavsiye edilir. ...

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<tr>
<td>New Item</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>L6876-1G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>24 well VDX Plate</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-142</td>
<td>&nbsp;</td>
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<td>24 well Cryschem Plate</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-158</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dow Corning Vacuum Grease</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-510</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Siliconized glass circle coverslides</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-231</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>100% paraffin oil</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-411</td>
<td>&nbsp;</td>
</tr>
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<td>1.88 inch wide Crystal Clear Sealing Tape</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-511</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>96 Well Imp@ct Plate (Microbatch plate)</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-098</td>
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protein crystallization for x-ray crystallography

Nasıl işlediğini anlaması için biyolojik makromoleküllerin üç boyutlu yapısını kullanarak, modern biyolojinin en önemli alanlarından biridir. Atomik çözünürlükte yapıların durumu derin ve benzersiz bir protein fonksiyonunun anlaşılmasını sağlar ve canlı bir hücrenin iç işleyişini aydınlatmak için yardımcı olur. Bugüne kadar,% 86 Protein Data Bank (rcsb PDB) girişleri makromoleküler yapıları X-ışını kristalografisi kullanılarak belirlenmiştir. Kristalografik çalışmalar için uygun kristaller elde etmek için, makromolekül (örneğin protein, nükleik asit, protein-protein kompleksi ya da protein, nükleik asit kompleksi) homojenlik arıtılmış olmalıdır, ya da mümkün olduğunca yakın homojenliği için. Hazırlık homojenliği (McPherson, 1999 Bergfors, 1999) yüksek çözünürlüklü metodla kristaller elde edilmesinde önemli bir faktördür. Kristalizasyon doygunluk makromolekül getiren gerektirir. Bu nedenle, örnek makromolekül (genellikle 2-50 mg / ml) agregasyonu veya yağış neden olmadan, mümkün olan en yüksek konsantrasyon konsantre olmalıdır. Presipite ajan örnek Tanıtımı çözüm büyüyen büyük üç boyutlu kristaller neden olabilir çözüm, protein kristalleri çekirdeklenme teşvik edebilir. Buharı difüzyon ve toplu kristalizasyon: kristaller elde etmek için iki ana teknikleri vardır. Buharı difüzyon, yağışlı ve protein çözümleri bir karışımını içeren bir damla saf Yağışlı bir odaya kapatılır. Damla ve yağışlı ozmolarite (Şekil 1A) eşit oluncaya kadar su buharı sonra açılan yayılır. Denge sağlanana kadar açılan dehidratasyon faz diyagramı ideal kristal çekirdeklenme bölgesi, hem de protein ve yağışlı yavaş bir konsantrasyon neden olur. Yağışlı (Şekil 1B) uygun miktarda protein ile karıştırma protein tarafından doğrudan çekirdeklenme bölgeye getirerek toplu yöntemi dayanıyor. Bu yöntem genellikle, açılan suyun yayılmasını önlemek için bir parafin / mineral yağ karışımı altında yapılır. Burada yağ altında toplu kristalizasyon ek olarak, damla ve oturma damla asılı buharı difüzyon için deney düzeneği iki türlü gösterecektir.

Crystallization is usually referred to as the bottleneck of X-ray crystallography. A sparse matrix incomplete factorial screen of precipitating conditions typically produces many different types of protein aggregation and precipitation, among them large single crystals. If the protein or precipitant concentrations are too high one can see brown matter with no distinct shape and size (amorphous precipitation). When the solution is undersaturated, the drop will often be completely clear and devoid of any kind of precipitat...

Watch this Scientific Journal Video about Protein Crystallization for X-ray Crystallography at JoVE.com

Protein Crystallization for X-ray Crystallography

3-D bir molekül yapısı molekül fonksiyonları nasıl eşsiz bir anlayış sağlar. Yakın atomik çözünürlükte yapı tespiti için temel yöntem, X-ışını kristalografisi. Burada, X-ışını kristalografisi yapı tayini için uygun olan herhangi bir makromolekül üç boyutlu kristaller elde etmek için mevcut yöntemler göstermektedir.

X-ray Kristalografi Protein Kristalizasyon

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

protein-crystallization-for-x-ray-crystallography

Research

JoVE Journal

Biology

63.0K Görüntüleme.  Yale University. 3-D bir molekül yapısı molekül fonksiyonları nasıl eşsiz bir anlayış sağlar. Yakın atomik çözünürlükte yapı tespiti için temel yöntem, X-ışını kristalografisi. Burada, X-ışını kristalografisi yapı tayini için uygun olan herhangi bir makromolekül üç boyutlu kristaller elde etmek için mevcut yöntemler göstermektedir.

Video: Protein Crystallization for X-ray Crystallography

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Sinkrotron-X-ray tomografi Sub-mikron Çözünürlük ile Non-invaziv 3D Görselleştirme

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Biyoloji

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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 ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

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 ...

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 ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

Preklinik X-ışını bilgisayarlı tomografi Veri 3D Yazdırma Setleri

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Rastgele Microseed Matrix ELEME Protein Belirginleşmesine Başarı Oranı artırma

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

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 ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Verimli memeli hücre ekspresyon ve Kristalizasyon için hücre dışı Glikoproteinlerin Tek aşamalı saflaştırma

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...