Biz bazı yöntemleri ile ilgili deneyim ve gözlemler katkıda bulunan değerli protein örnekleri, sağlamak için bizimle işbirliği yapanlara teşekkür ediyorum. Günther Schmalzing lütfen bu projeye katılmaya FR için fırsat sağladı. Barbara Armbruster, Jacob Brink ve Deryck Mills olağanüstü yardım ve ekipman giriş için teşekkür. Finansman NIH hibe HL090630 tarafından sağlanmıştır.

1. 2B Kristalizasyon Denemeler Izgara Hazırlama <ol><li> Karbon-400-mesh kaplı bakır EM ızgaraları negatif leke tarafından hazırlanmıştır. Uranil asetat sık kullanılan ve ızgaraları uzun süreli depolama için kullanımının yanı sıra uygunluğu önce birkaç ay boyunca uzun süreli bir çözüm depolama açısından leke sağlar. Buna karşılık, uranil format gibi diğer olumsuz lekeleri, mükemmel boyama sağlarken, taze 1 yapılabilir. Izgaraları 2D kristalizasyon çalışmaların tarama için kullanılmak üzere çok sayıda hızlı bir şekilde hazırlanması için, negatif boyama değiştirilmiş bir versiyonu kullanılmıştır. 2 mcL örnek hacmi, bir karbon kaplı EM ızgara üzerine pipetlenebilir ve 60 saniye süreyle inkübe Bu whatman bir yırtık parça # 4 filtre kağıdı (Şekil 1; video) kenarından blot takip eder ve daha sonra% 1&#39;lik uranil asetat 2 mcL hemen uygulanır, 30 sonra tekrar ızgara kenarından kurutulur s Filtre kağıdı yırtılmış kenarı ile jant üzerinde grid dokunmak proteoliposomes kaldırılması olmadan sıvı optimal çıkarılması sağlar. Ayrıca, ızgara kenarında kurutma, karbon film geliştirilmiş koruma sağlar. Hassas karbon film kırılması örnek yapışmayı önler ve örnek bir yanlış temsili neden olabilir Bakım, hazırlama ve ızgaraları işleme alınması gerekir. 5 mcL geleneksel büyük örnek hacmi ve ızgara hazırlanması için rutin olarak kullanılan iken, değerli örnekleri 2 mcL veya daha az 2 hacmi azaltarak kaydedilebilir . </li><li> Mümkün olan en yüksek membranlar üretmek için, bazı örneklerin gliserol veya sukroz, yüksek konsantrasyonlarda (% 10-20), diyaliz tampon mevcut olması gerekir. Gliserol veya sukroz, yüksek viskoz ve uranil asetat tampon düzgün bir nüfuz ve böylece eksik membranlar boyama engeller, ızgara hazırlanması üzerinde olumsuz bir etkisi olabilir. Dolayısıyla olası bir kafes gizlenmiş olacaktır. Ya tampon, gliserol / sakaroz-tampon (gösterilmemiştir), veya ızgaraları ve yedek örnekleri santrifüj ile değiş tokuş edilebilir benzer negatif boyanma önce birkaç döngüleri tampon ya da tek bir gliserol / sakaroz ücretsiz tampon yıkanabilir cryo-EM 3,4 için kullanılan bir tekniktir. </li></ol> 2. EM 2D Kristalizasyon Denemeler Değerlendirilmesi <ol><li> Numune sahibinin türüne bağlı olarak, bir veya birden fazla ızgaraları ya yüklenir. Burada ara büyütme sevk edilecektir 2-10K, büyütme, laboratuvar dizüstü 5 not alınır membranlar, morfolojisi ve büyüklüğü, dağılımı ortalama dağılımı ve kapsamı bir ilk izlenim sağlar. CCD kamera, film üzerinde mevcut değilse, uygun alanlarda bir CCD kamera yardımıyla temsili Özetler olarak kaydedilir veya. </li><li> Tüm örnek / ızgara bir bakış istendiğinde kabaca 400-800x aralığında düşük büyütme, zaman zaman kullanılır. Her ızgara ile istihdam olmasa da, düşük büyütme ızgara hazırlık çeşitli yönleri açısından değerlendirilmesinde yardımcı değerli bilgiler verir: negatif boyama ve karbon film, ızgara üzerinde örnek konsantrasyon, ve muhtemelen düzensiz proteoliposome dağıtım potansiyel kısmi kırılması. Bireysel ızgara kareler dürbün veya CCD kamera ile görüntülenebilir olabilir. Bazı sabahları yüksek büyütmelerde daha sonra muayene için geri çağrılabilir ve özellikle ilgi çekici pozisyonları, kaydetmek mümkündür. </li><li> Ilgi ızgara alanı, ya düşük veya orta büyütme tespit edildikten sonra, büyütme yaklaşık 50K-60k değiştirildi. 30K olarak düşük ve 80K gibi yüksek büyütmeler, eğer biliniyorsa, membran, kristal ve birim hücre boyutuna bağlı olarak kullanılır. 30-80K büyütme aralığı 2D kristalleri için tarama amacıyla yüksek büyütme olarak sevk edilecektir. Odaklama, resim / fotoğraf ayarı, ya da ilgi alanı çevresinde sonraki anahtarı ile odak ayarı düşük doz set-up ya da oluşur. <ol class="lalpha"><li> Gerçekten bir zar ilgi alanı olup olmadığını belirsizlik durumlarında, örnek proteoliposomes, mika, ya da diğer eserler büyüklüğü, karbon film için denetlenir. Bu amaçla, kenarlar tipik bir katlama ve morfoloji ortaya koymaktadır. </li><li> Şimdi ilgi alanında bir CCD kamera ile denetlenir. CCD görüntü 30K-80K büyütme toplanan membran boyutu, protein veya birim hücre boyutunu bağlı olarak, ya da kristal alan bilinmektedir. Küçük ve / veya çoğunlukla hidrofobik membran proteini kafes CCD görüntü kendisi görsel değerlendirme mutlaka görünmez. Ya görüntünün tamamını online Fourier dönüşümü (FT, ya da hızlı FT-FFT) kullanılır. Bu FT sipariş dizi küçük ise, ancak önemli bir miktar gürültü içerecektir. Böylece, bir simgesi ve gelişmiş bir sinyal-gürültü oranı düşük bir kutulu görüntü boyutu için izin verecekler kristal ve kolay tanımlama. Bu amaçla, kutu resmin üzerine taşınır ve canlı bir FT değerlendirilir. Kiriş / parlaklık yoğunluğu düşük doz örnek için koşulların yanı sıra akılda CCD kamera ayarları tutmak ayarlanır. Kullanılan CCD kamera bağlı olarak, canlı FT gama sipariş dizilerin en iyi tanımlanması için ayarlanır. Aşırı yüksek bir değer gürültü katkıları nedeniyle noktalar belirsiz olabilir ve aşırı derecede düşük bir gama değeri zayıf noktalar tespit ediliyor önleyecektir. Bu zayıf noktalar, ancak gürültü seviyesinden FT noktalar ile küçük kristal dizileri nedeniyle olabilir. Örnekleri 6, az sayıda yüksek çözünürlükte veri toplanmış olsa da, olumsuz lekeli 2D kristalleri yaygın çözünürlük ile sınırlı ya da kabaca 15A çözünürlük daha iyi olması beklenmemelidir. Ile yaklaşık -400 nm bulanıklaştırma, lekeler fazla 1-3 siparişleri kolayca tanınabilir olması bekleniyor. Örnekleri cryo-EM veri toplama, en ulaşılabilir çözünürlüğü uygun bir göstergesi olarak, genellikle çözümü için değerlendirilir değildir. Noktalar ve olası mosaicity Netlik olsa belirtilmiştir. </li></ol></li><li> Farklı membranlar, membran morfolojileri yanı sıra, yüksek büyütme sipariş için değerlendirilir. Bu, küçük değil, büyük proteoliposomes veya membran yamaları en çok gelecek vaat eden alanlar içermesi gibi, başında veya 2D kristalizasyon çalışmaların ara aşamadan özellikle kritik. 2B kristalleri çok düşük yüzdeler sipariş dizilerin ilk kimlik boyutu ve kalitesi 2,4,7 hızlı bir gelişme sık sık neden bu görüntülerin çok sayıda görüntü elde etme ve FT, ya da optik kırınım, gerektirecektir . </li></ol> 3. Temsilcisi Sonuçlar İdeal sipariş proteoliposomes kolayca tanınabilir, keskin noktalar gösterir. Geniş ve iyi düzenlenmiş kristaller kolayca CCD görüntü veya mikrograflar optik kırınım online-FT tarafından tespit edilir. Örneğin, küçük boyutta birkaç mikron kadar 18kDa bir membran proteini 2D kristalleri gösterir. FT lekeler kolaylıkla tespit ve keskindir. Canlı FT kutusu hareket kafes mosaicity olmadan sürekli olduğunu gösterir. EM küçük ekran, daha geniş bir çözünür etki alanı ile daha büyük bir protein kafes tespit edilebilir. CCD görüntü toplama ve FT, daha iyi değerlendirilmesi için bir araç sağlamak ve, örneğin, mümkün mosaicity (gösterisi FT) hakkında bilgi elde etmek için gerekli. Sıralı olmayan bir proteoliposome FT hesaplanırken, gürültü başlangıçta noktalar için yanlış olabilir. Canlı-FT kutu hareket olmasına rağmen, ancak, lekeler kaybolur. Öte yandan, küçük diziler, şüpheli kristallik, canlı-FT hatta biraz görüntü alana taşındığında sabit kalan noktalar olacaktır. Ayrıca, bu küçük kristaller, genellikle aynı birim hücre boyutları olan tarafından kabul edilebilir ve farklı FTS noktalar arasındaki mesafeler, belirli bir büyüklükte bir daire gibi çeşitli şekillerde ölçülebilir. Lipid kristaller ayrı bir kafes morfolojisi ve FT gösterir. Ilk çalışmalarda yağış karşılaşmak nadir değildir. Sulandırıldıktan olmadan protein yağış olsa küçük lipid agrega ayırt edilmesi gerekiyor. Düşük büyütmede çökeltileri olarak görünür Örnekleri sık sık yüksek büyütmede bakıldığında lipid agrega çıkıyor. 30-50K inceleme üzerine, bu karanlık yapıların köşeleri, onları hiç yağış protein ile membran oluşur ortaya koymaktadır. Aşağıdaki deneyler büyük zarlarında lipid agrega boyutu artmış olabileceği gibi, bu önemli gözlemler. Değerlendirirken örnekleri Kötü sonuçlar bazen doğru ve hızlı bir tarama önleyen düşük bir membran konsantrasyonuna bağlı. Bu durum sıklıkla diyaliz ile 2B kristalizasyon için yüksek protein konsantrasyonu kullanımı ile aşılabilir. Alternatif olarak, membranlar Eppendorf tüp depolama sırasında alt kısmında bir kaç gün dinlenmeye bırakıldı. Bazı durumlarda membran hızlı, ya da neredeyse anında yerleştikten oluşur ve tüpün dibinden pipetleme ızgara daha yüksek bir membran yoğunluğu neden olacaktır. Çok daha hızlı bir başka seçenek tüp alttan sonraki örnekleme ile örneklerinin santrifüj (1-3 dakika 3000-8000 rpm). Optimal koşullarda Örnekleri 2D kristallerinin büyük bir yüzdesini içerecektir. Bu en büyük ve en iyi sipariş 2D kristalleri veriler için görsel olarak seçilen topluluğu olarak, membran homojen bir görünüm için amacı gerekli değildir. Kristalizasyon denemeler gibi örnekler için kullanıldığı zaman tekrarlanan bu tür örnekleri kolayca tanınırmaksimum sayıda yüksek çözünürlüklü görüntüleri ile sonuçlanan cryo-EM veri toplama,. <img src="/files/ftp_upload/1846/1846fig1.jpg" alt="Şekil 1" /> Şekil 1: Bu rakam whatman # 4 filtre kağıdı yırtılmış bir parça ızgara kenar blot gösterir.

<ol>
	<li>Johansen, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+field+electron+microscopy+of+biological+specimens+V.+A+low+dose+pre-irradiation+procedure+reducing+beam+damage.">Bright field electron microscopy of biological specimens V. A low dose pre-irradiation procedure reducing beam damage.</a> Micron. 7, 145-156 (1976).</li><li>Schmidt-Krey, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+crystallography+of+membrane+proteins:+Two-dimensional+crystallization+and+screening+by+electron+microscopy.">Electron crystallography of membrane proteins: Two-dimensional crystallization and screening by electron microscopy.</a> Methods. 41, 417-426 (2007).</li><li>Wang, D. N., K&#252;hlbrandt, W. <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+electron+crystallography+of+light-harvesting+chlorophyll+a/b-protein+complex+in+three+different+media.">High-resolution electron crystallography of light-harvesting chlorophyll a/b-protein complex in three different media.</a> J Mol Biol. 217, 691-699 (1991).</li><li>Schmidt-Krey, I., Rubinstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryomicroscopy+of+membrane+proteins:+specimen+preparation+for+two-dimensional+crystals+and+single+particles.">Electron cryomicroscopy of membrane proteins: specimen preparation for two-dimensional crystals and single particles.</a> Micron. , (2010).</li><li>Schmidt-Krey, I., Mutucumarana, V., Haase, W., Stafford, D. W., K&#252;hlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+of+human+vitamin+K-dependent+&amp;gamma;-glutamyl+carboxylase.">Two-dimensional crystallization of human vitamin K-dependent &amp;gamma;-glutamyl carboxylase.</a> J Struct Biol. 157, 437-442 (2007).</li><li>Trachtenberg, S., DeRosier, D. J., Zemlin, F., Beckmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-helical+perturbations+of+the+flagellar+filament:+Salmonella+typhimurium+SJW117+at+9.6+&amp;Aring;+resolution.">Non-helical perturbations of the flagellar filament: Salmonella typhimurium SJW117 at 9.6 &amp;Aring; resolution.</a> J Mol Biol. 276, 759-773 (1998).</li><li>Zhao, G., Johnson, M. C., Schnell, J. R., Kanaoka, Y., Irikura, D., Lam, B. K., Austen, K. F., Schmidt-Krey, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+conditions+of+human+leukotriene+C4+synthase+requiring+a+particularly+large+combination+of+specific+parameters.">Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring a particularly large combination of specific parameters.</a> J Struct Biol. 169, 450-454 (2010).</li><li>Cheng, A., Leung, A., Fellmann, D., Quispe, J., Suloway, C., Pulokas, J., Abeyrathne, P. D., Lam, J. S., Carragher, B., Potter, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+automated+screening+of+two-dimensional+crystals.">Towards automated screening of two-dimensional crystals.</a> J Struct Biol. 160, 324-331 (2007).</li><li>Vink, M., Derr, K. D., Love, J., Stokes, D. L., Ubarretxena-Belandia, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+strategy+to+screen+2D+crystallization+trials+of+membrane+proteins.">A high-throughput strategy to screen 2D crystallization trials of membrane proteins.</a> J Struct Biol. 160, 295-304 (2007).</li></ol>

Uygun değerlendirme örneklerinin membran yeterli sayıda dikkatli bir değerlendirme gerektirir. Örneğin, 180 üzerinden görüntülü proteoliposomes düşük% 2 olarak kristal dizileri örnekleri 2D kristalizasyon koşullarına hızlı optimizasyon 7 için kritik bilgi verdi. Zaman zaman kısmi protein yağış görülse de, bir ızgara protein yağış oluştuğunda, düşük büyütmede incelemeden sonra, daha fazla tarama terk olabilir. Olsa bile çok az 100 nm küçük m...

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<td>400-mesh copper TEM grids coated with carbon film</td>
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<td>forceps: regular and anti-capillary </td>
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<td>Dumont #5 and Dumont N5AC or similar</td>
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<td>Micropipette and pipette tips</td>
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<td>Whatman #4 filter paper</td>
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<td>1% uranyl acetate</td>
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<td>Dialysis sample to be screened for 2D crystals</td>
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<td>Glycerol/sucrose-free dialysis buffer</td>
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<td>Optional</td>
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<td>JEOL-1400 transmission electron microscope (TEM)</td>
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<td>similar 80 &#x2013; 120kV TEM equipped with an Lab6 or tungsten filament and film and/or CCD cameras (Gatan Orius SC1000 and/or UltraScan1000 CCD cameras and Gatan Digitial Micrograph software package or Tietz cameras (TVIPS)) </td>
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assessing two-dimensional crystallization trials of small membrane proteins for structural biology studies by electron crystallography

Elektron kristalografisi, ya alternatif olarak veya membran proteinlerinin yapı-işlev sorular, eriyen proteinlerin yanı sıra çalışma, üç boyutlu kristalizasyon ve X-ışını kristalografisi birlikte kullanılabilecek bir yöntem olarak gelişti. Transmisyon elektron mikroskobu ile iki boyutlu (2D) kristalleri için tarama (EM) optimize ve cryo-EM tarafından yüksek çözünürlüklü veri toplama için örnekler seçerek bulma kritik bir adımdır. Burada potansiyel kristalizasyon koşullarının optimizasyonu için kritik bilgi kaynağı olabilir, büyük ve sipariş yanı sıra küçük 2D dizileri belirlenmesinde temel adımları açıklar. EM farklı büyütme ile çalışarak, bir dizi kritik parametreleri veri elde edilir. Alt büyütme, morfolojisi ve membran boyutu değerli veri sağlamaktadır. Yüksek büyütmeler, düzen ve 2D kristal boyutları belirlenir. Bu bağlamda, yüksek büyütmelerde kullanılan CCD kamera ve online-Fourier Dönüşümleri düzen ve boyutu için proteoliposomes değerlendirmek için nasıl tarif edilir. Membran proteinlerinin 2D kristalleri diyaliz ile sulandırıldıktan en yaygın olarak yetiştirilen, tarama tekniği, mono tabakaları, yerli 2D kristaller yardımıyla üretilen kristal için de geçerlidir ve çözünür proteinler dizileri emretti. Buna ek olarak, burada açıklanan yöntemleri büyük proteinler ve bizim örnek ve kafes gibi küçük proteinlerin belirlenmesinde aynı miktarda bakım gerektiren daha küçük 2D kristalleri gibi büyük membran proteinleri, tarama daha kolay tanımlanabilir olabilir tarama önceki aşamada.

Watch this Scientific Journal Video about Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography at JoVE.com

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

Elektron kristalografisi sipariş membran protein dizileri oluşumu için iki boyutlu (2D) kristalizasyon çalışmalarda değerlendirilmesi son derece kritik ve zor bir iştir. 90kDa - Burada tarama ve 15 aralığında ağırlıklı olarak küçük membran proteinlerinin 2D kristalleri tespit yaklaşım tarif.

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 ...

assessing-two-dimensional-crystallization-trials-small-membrane

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Biology

11.2K Görüntüleme.  Georgia Institute of Technology. Elektron kristalografisi sipariş membran protein dizileri oluşumu için iki boyutlu (2D) kristalizasyon çalışmalarda değerlendirilmesi son derece kritik ve zor bir iştir. 90kDa - Burada tarama ve 15 aralığında ağırlıklı olarak küçük membran proteinlerinin 2D kristalleri tespit yaklaşım tarif.

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

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

Proteinlerin Hücre Biyolojisi Microcontact Baskı

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Biyoloji

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 ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

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 ...

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 ...

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 ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Verimli Üretimi ve Saflaştırılması Rekombinant murin kindlin-3 Biophysical Çalışmaları Böcek hücrelerinden

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

Elektron Cryo-tomografi ile Mitokondri ATP Sintazı dimerlerini Görselleştirme

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

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.