我々は我々の方法に関連する経験と観察のいくつかに貢献し、貴重なタンパク質サンプルを、提供するための私達の協力者に感謝する。ギュンターSchmalzingは親切にこのプロジェクトに参加するFRする機会を提供した。バーバラArmbruster氏、ヤコブブリンクとデリックミルズは、その卓越したヘルプや機器の入力のために感謝されています。資金は、NIHの助成金HL090630によって提供されていました。

1。二次元結晶化トライアルのグリッドの準備<ol><li>炭素でコーティングされた400メッシュ銅EMグリッドは負染色することによって調製される。酢酸ウラニルは、頻繁に使用され、長期的なグリッドの長期保存のための使用だけでなく、適合性の前に数ヶ月のためのソリューションのストレージの条件で染色を提供しています。対照的に、このようなウラニルギなどの他の負の汚れ、優れた染色を提供している間は、新鮮な1を行う必要がある。二次元結晶化試験のスクリーニングに使用されるグリッドの多数の高速な準備作業については、ネガティブ染色を変更したバージョンが使用されます。サンプル2μLの体積は、炭素で覆われたEMのグリッド上にピペットで、60秒間インキュベートする再び30の後、グリッドの端からブロットされ、直ちに適用され、その後、1％酢酸ウラニルの2μL、これをWhatman＃4ろ紙（ビデオ図1）の破れた部分とエッジからブロッティングが続きます。 S.ろ紙の破れたエッジとリム上のグリッドに触れると、プロテオリポソー​​ムを除去することなく液体の最適な除去を保証します。さらに、グリッドの端に乾燥には炭素膜の改良維持を保証。繊細な炭素膜の破損がサンプルの付着を防止し、サンプルの不正確な表現につながることができるようにケアは、準備とグリッドの取扱いに注意する必要があります。 5μLの伝統的に大規模なサンプルの量があったと日常的にグリッドの準備のために使用されていますが、貴重なサンプルを2μLまたは2以下にボリュームを削減することで保存することができます。 </li><li>可能な限り最大の膜を生成するために、我々のサンプルのいくつかは、グリセロールまたはショ糖の高濃度（10-20％）が透析緩衝液中に存在する必要があります。グリセリンまたはスクロースが高粘度であり、適切にバッファを貫通するから酢酸ウラニルを防ぐため、不完全膜を染色するので、これは、グリッドの準備にマイナスの影響を持つことができます。したがって可能な格子が不明瞭になります。バッファーはグリセロール/ショ糖を含まないバッファ（図示せず）によってサンプルと交換の遠心分離によって交換することができる、またはグリッドが似てネガティブ染色の前に数サイクルに1つのバッファまたはグリセロール/ショ糖を含まない緩衝液で洗浄することができるのどちらかクライオEMは3,4で使用される手法。 </li></ol> 2。 EMによる二次元結晶化トライアルの評価<ol><li>試料ホルダーのタイプに応じて、いずれかまたは複数のグリッドがロードされます。中間倍率としてここに呼ばれる2 - 10K、の倍率は、実験ノート5の中のメモを取られている膜、形態、サイズ、の分散の平均の分布と程度の第一印象が可能になります。 CCDカメラはフィルムで、利用できない場合、適切な領域は、CCDカメラの助けを借りて、代表的な概要として記録またはされています。 </li><li>約400 - 800 ×の範囲の低倍率は、全体のサンプルの概要/グリッドが望まれる時間に使用されます。両方否定的染色およびカーボン膜、グリッド上のサンプルの濃度、および場合によっては不均一なプロテオリポソー​​ム分布の潜在的な部分的な破損：すべてのグリッドで採用されていないが、低倍率では、いくつかの側面の面でグリッドの準備の評価の助けの貴重な情報を提供します。個々のグリッドの四角形は、双眼鏡やCCDカメラで表示される場合があります。一部のEMSとは、より高い倍率で、後で検査のためにリコールすることができる特定の興味のあるポジションを、保存することが可能です。 </li><li>興味のグリッド領域が低いか中間倍率で識別されると、倍率は約50K - 60Kに変更されます。膜、水晶とユニットセルのサイズに応じて、既知の場合、として30Kと低いと80Kという高倍率が使用されている。 30 - 80Kの倍率の範囲は、2次元結晶のスクリーニングの目的のために高倍率と呼ぶことにする。焦点は、低用量のセットアップ、画像/写真の設定に続いてスイッチ付き、または関心のある領域の近傍でのフォーカスの設定のいずれかで発生します。 <ol class="lalpha"><li>関心の領域が実際に膜であるかどうかのあいまいさのケースでは、サンプルはプロテオリポソー​​ムの大きさの炭素フィルム、マイカ、またはその他の成果物のために検査されます。この目的のために、エッジは典型的な折りたたみや形態を明らかにする。 </li><li>今関心の領域がCCDカメラで検査されます。 CCDイメージは30K - 80K膜のサイズに応じて倍率、、タンパク質または単位セルサイズ、または既知の結晶性の領域で収集されます。小規模および/または主に疎水性の膜タンパク質の格子は、CCDの画像自体の視覚的評価では必ずしも表示されません。どちらかの全体像は、オンラインフーリエ変換（FT、または高速のFT - FFT）に使用されます。順序付き配列が小さい場合は、このFTは、しかし、ノイズのかなりの量が含まれます。このように、縮小箱入り画像サイズはSmaIでの改善された信号対雑音比が可能になりますLER結晶と容易に識別。この目的のために、ボックスは画像の上に移動され、ライブFTが評価されます。 ビームの強度/輝度は低用量サンプルの条件だけでなく、心のCCDカメラの設定を維持する調整されます。使用されるCCDカメラによっては、ライブFTのガンマは、順序付けられた配列の最適な識別のために調整されます。過度に高い値は、ノイズの寄与に起因するスポットを隠蔽したりすることができます、そして過度に低いガンマ値が識別されるから弱いスポットを防止します。これらの弱い点はほとんどノイズレベルよりFTの斑点のある小さな結晶配列が原因である可能性があります。 より高い分解能でデータをサンプル6の少数の収集されているが、一般的にネガティブ染色2D結晶の分解能に制限されたり、およそ15Aの解像度よりも優れていると想定してはいけません。約-400 nmのデフォーカスで、斑点のない以上の1から3までの注文が簡単に識別できることが期待されています。サンプルは一般的に低温電子顕微鏡データの収集が達成可能な最高解像度の適切な指示を与えるとして、解決のために評価されません。スポットと可能mosaicityのシャープネスはも記載されています。 </li></ol></li><li>異なる膜だけでなく、膜の形態は、高倍率での順序のために評価されます。小さいのではなく、大きなプロテオリポソー​​ムまたは膜のパッチが最も有望な分野を含むことができるので、これは、最初または2次元結晶化試験の中間段階では特に重要です。 2次元結晶の非常に低い割合では、順序付けられた配列の最初の同定以来、多数の画像の画像取得とFT、または光学回折は、頻繁にサイズと品質2,4,7の急速な向上につながることが必要になります。 </li></ol> 3。代表的な結果理想的に順序付けられたプロテオリポソー​​ムは容易に認識可能な、シャープなスポットを表示する。大と規則正しい結晶は容易にCCDの画像や顕微鏡写真の光学回折のオンライン- FTによって識別されます。 例のサイズは数ミクロンまでである18kDaの小さな膜タンパク質の2次元結晶を示しています。 FT上のスポットを簡単に識別し、シャープされています。ライブ- FTのボックスの動きは、格子がmosaicityなしで連続であることを示しています。より広範な可溶性ドメインを持つ大規模タンパク質の格子は、EMの小さな画面上で識別することができます。 CCDイメージコレクションとFTは、より良い評価の手段を提供すると、例えば、可能な限りmosaicity（ショーFT）の情報を得ることが必要である。順序付けされていないプロテオリポソー​​ムのFTを計算する場合、ノイズは、最初のスポットと誤解することができます。ライブ- FT用のボックスが移動させながら、しかし、斑点が表示されなくなります。一方、小規模のアレイは、不審な結晶で、ライブFTが画像領域上にもわずかに移動するときに静止して、残りの自分の斑点を持つことになります。さらに、これらの小さな結晶は、通常、同一のユニットセルサイズを持つことによって認識することができる、と別のFTSでスポット間の距離は、特定の大きさの円を持つなど、様々な方法で測定することができます。脂質の結晶は、異なる格子の形態とFTが表示されます。 それは初期の試験で降水量が発生することも珍しくありません。ここに再構成せずにタンパク質沈殿はいえ、小さな脂質凝集体と区別する必要があります。低倍率で析出されているように見えるサンプルは、頻繁に高い倍率で見た場合の脂質凝集体であることが判明する。 30 50Kでの検査時には、これらの暗い構造のエッジは、それらが蛋白質の無降水量を有する膜で構成することが明らかに。脂質凝集体は、以下の実験で大規模な膜にサイズを大きくする可能性があるので、これらは重要な観察である。 サンプル評価の悪い結果は、しばしば適切かつ高速なスクリーニングを防ぐ低膜濃縮に接続されています。これは、頻繁に透析の2次元結晶化のための高タンパク質濃度を使用することで克服することができる。また、膜は、貯蔵中のエッペンドルフチュー​​ブの下部に数日間のために解決するために残すことができます。いくつかのケースでは膜の急速な、またはほとんどインスタントセトリングが発生し、チューブの底からペッティングすれば、グリッド上でより高い膜密度になります。別のはるかに高速のオプションは、チューブの底からの後続のサンプリングとサンプルの遠心分離（1〜3分間3000から8000 rpmで）です。 最適な条件下でのサンプルは、2次元結晶の大部分が含まれます。それは最も大きく、最もよく秩序2D結晶がデータ収集のために視覚的に選択されているように、膜の均質な外観を目指す必要はありません。サンプルはのために使用されている場合、結晶化試験は同様に繰り返されている場合、サンプルのこれらのタイプは簡単に認識されます。高解像度の画像の最大数は、結果として低温電子顕微鏡データの収集、。 <img src="/files/ftp_upload/1846/1846fig1.jpg" alt="図1" /> 図1この図は、ワットマンの破れた部分第4濾紙でグリッドの端をブロッティングを示しています。

<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>

サンプルの適切な評価は、膜の十分な数の慎重な評価が必要です。例えば、180以上の画像化されたプロテオリポソームの外のような低％2などの結晶配列を持つ試料は二次元結晶化の条件7の迅速な最適化のための重要な情報を与えた。 タンパク質の時折部分的沈殿が発生したものの、タンパク質の沈殿が発生すると、グリッドは、低倍率での検査の後、さらにス?...

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<td>400-mesh copper TEM grids coated with carbon film</td>
<td>&nbsp;</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>
<td>&nbsp;</td>
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<td>Whatman #4 filter paper</td>
<td>&nbsp;</td>
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<td>1% uranyl acetate</td>
<td>&nbsp;</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

電子線結晶学は、どちらかあるいは、または構造と機能膜タンパク質の質問だけでなく、可溶性タンパク質を研究するために三次元結晶化とX線結晶構造解析と組み合わせて使用​​することができる手法として発展してきた。透過型電子顕微鏡による二次元（2D）の結晶のスクリーニングは、（EM）を最適化し、低温電子顕微鏡による高分解能のデータ収集のためのサンプルを選択し、見つけるのに重要なステップです。ここでは、潜在的に結晶化条件の最適化に重要な情報を供給できるよう、大規模および順序だけでなく、小規模な2次元配列の両方を識別するための基本的な手順を説明します。 EMで異なる倍率で作業することによって、重要なパラメータの範囲のデータが得られる。低倍率では、形態や膜サイズで貴重なデータを提供しています。より高い倍率で、可能な限り順番と二次元結晶の寸法が決定されます。この文脈では、CCDカメラとオンライン - フーリエ変換は、順序とサイズのためのプロテオリポソー​​ムを評価するために高倍率で使用されているか説明しています。 膜タンパク質の2次元結晶は、最も一般的に透析して再構成によって栽培されているが、スクリーニングの手法は、単層、ネイティブの2Dクリスタルの助けを借りて生産結晶の等しく適用可能である、と可溶性タンパク質の配列を命じた。さらに、ここで説明する方法は、さらに小さいの2D結晶だけでなく、私たちの例と大規模なタンパク質の格子がより容易に識別できる可能性があるとしても小さい蛋白質が同定におけるケアの同じ量を必要とする大規模な膜タンパク質のスクリーニングに適用可能である審査の早い段階で。

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

注文した膜タンパク質アレイの形成のために2次元（2D）の結晶化試験を評価すると、電子線結晶学における非常に重要かつ困難な作業です。 90kDa - ここでは、のためにスクリーニングし、15の範囲で主に小型の膜タンパク質の2次元結晶の同定に我々のアプローチを説明します。

電子線結晶学による構造生物学研究用小型膜タンパク質の二次元結晶化トライアルの評価

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

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JoVE Journal

Biology

11.2K ビュー.  Georgia Institute of Technology. 注文した膜タンパク質アレイの形成のために2次元（2D）の結晶化試験を評価すると、電子線結晶学における非常に重要かつ困難な作業です。 90kDa - ここでは、のためにスクリーニングし、15の範囲で主に小型の膜タンパク質の2次元結晶の同定に我々のアプローチを説明します。

ビデオ: 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.

細胞生物学のためのタンパク質のマイクロコンタクトプリンティング

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

生物学

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.

脂質中間相を用いた構造決定のための膜タンパク質の結晶化

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 -線結晶構造のためのタンパク質の結晶化

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.

脂質中間相における膜タンパク質の結晶化

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.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

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.

脂質中間相における膜タンパク質のハイスループット結晶化のためのロボットの使用

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.

高分子結晶学による構造決定のための脂質メソフェーズで栽培膜タンパク質の収穫とクライオ冷却結晶

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.

生物物理学研究のための昆虫細胞からの効率的な生産と組換えマウスKindlin-3の精製

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.

電子クラ​​イオトモグラフィーによるミトコンドリアにおけるATP合成酵素二量体の可視化

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.