この作品は、YMにバロウズウェルカムの研究者賞で、エール大学からMDへのブラウン - コックスポストドクトラルフェローシップでサポートされていました。

材料： <ul><li>タンパク質試料 - リゾチーム（50 mg / ml）を</li><li>ドロップ24ウェルトレーハンギング</li><li>シッティングドロップ24ウェルトレー</li><li> Microbatchの結晶化96ウェルトレー</li><li>結晶化ソリューション（どちらか市販または自家製） </li><li>シリコングリース</li><li>ルアーロックせずに5 mLの注射器</li><li>シリコーンカバースライド</li><li>光学シールテープ</li><li>パラフィン</li><li>低保持のヒントと0.1〜2μLマイクロピペット</li><li>ピンセット</li><li>プロのワイプ</li></ul> 1。ハンギング/ドロッププロシージャを座って。 <ol><li>初期画面のために一つの試験条件のスパース行列不完全な階乗のメソッドを利用する市販のスクリーンを使用することができます。スパース行列の不完全要因加算法は、電圧値と高分子のための既知の結晶化条件（カーターとカーター、1979; Cudney ら、1994;。Jancarikと金、1991;。Jancarikら 、1991）から選択される。結晶化条件がすでにわかっている場合は、狭い画面はオリジナルのヒットの周りに沈殿剤濃度とpHを変化させること、組み立てることができます。最も一般的に成功した沈殿は硫安が続くポリエチレングリコール（PEG）、である。結晶化に使用したすべての記録された高分子の沈殿剤の約60％をこれら2つの沈殿剤は、アカウント（。ギリランドら 、1994;キンバーら、2003;。。らページ、2003）。 </li><li>あなたの結晶化溶液を調製。通常、これらの条件は、沈殿剤、ある種の塩とpHを設定するには、バッファから結合されます。場合によっては添加剤は、目的のタンパク質のために特別に追加される可能性があります。すべてのストック溶液を0.22μmのフィルターを使用してフィルタリングする必要があります。ストック溶液の一部は、（50％PEGは、グリセリン、PEG - MME、など）非常に粘性のあるかもしれない。 <ul><li> 0.6 1.5 mLの、0.8、1.0、1.2、1.4、0.1 M NaOAc pHは4.0、4.3、4.6と4.9の1.6 M NaClをそれぞれを（24ソリューションの合計）の準備</li></ul></li><li>あなたのタンパク質サンプルを解凍し、氷上に置きます。目的のタンパク質は、（もしそうなら、4℃で保存）/解凍サイクルを凍結する敏感でない限り、タンパク質の株式は、-80℃で保存してください。典型的なタンパク質の濃度は高濃度が通常より良い結果を与えることで、50〜50 mg / mLの範囲内である。 </li><li> 000xg / 4、サンプル15min/18をスピン° C - いくつかのタンパク質は、解凍工程の後に部分的に集約し、沈殿する傾向がある。これらの凝集体は、したがって、完全なスピンがダウンして、あなたのサンプル中の唯一の可溶性分子を持って確保するタンパク質分子の残りの非晶析出を促進することができます。トレイを設定するまで、氷上に蛋白質を保つ。 </li><li> UV分光光度計を用いて280nmでの吸光度からタンパク質濃度を決定する。タンパク質濃度は、吸光度測定値と蛋白質の吸光係数から計算することができます。 ：吸光係数はExpasyウェブサイト上のProtParamツールを使用して計算することができます<a href="http://ca.expasy.org/tools/protparam.html">http://ca.expasy.org/tools/protparam.html</a> </li><li> 24ウェルハング/トレイの設定のスキーム（図2B）によると沈殿剤溶液500μLをドロップトレイに座っての井戸を埋める。ウェルの縁の周りにシリコーングリースのリングを作成します。リングは良くはシールされているように空気の圧力上昇を防ぐための小さなギャップを持つ必要があります。座っ滴で、よくは（図2A）テープで封印されているため、グリースの必要はありません。 </li><li>カバースライドを取り、凝縮した空気スプレーで掃除、またはワイププロ - ダストが結晶化ドロップの解釈を容易にし、汚染を除去する避ける。ドロップ、カバースライドを座ってするために使用されていません。その代わり、よくは、タンパク質と沈殿剤とを混合されている棚を含んでいます。 </li><li>カバースライドでタンパク質試料とリザーバー溶液の等量を置く。タンパク質と沈殿剤の異なるボリュームを最適化プロセスの一部として試されることがあります。希薄なタンパク質サンプルのために望ましいことができ、結晶の核生成や成長を遅らせるために平衡後降下、でタンパク質の正味濃度の沈殿剤の結果よりも多くのタンパク質を使用する。ドロップソリューション内の他の不揮発性の物質も、同じ要因によって濃縮される。カバースライド上にタンパク質とリザーバー溶液をピペッティングするときに、気泡の形成を避けるために非常に注意してください。これは多くの場合空気​​がピペットから吹き出される場合に発生します。 <ul><li>カバーのスライドの中央に0.02 M NaOAcのpHを4.9に50 mg / mLのリゾチームのロード2μLをし、そのためにリザーバー溶液2μLを加える。懸滴。 </li><li>ドロップ座り：荷重棚の中心から50 mg / mLのリゾチーム溶液2μLをし、そのためにリザーバー溶液2μLを加える。 </li></ul></li><li>静かにカバーをスライドして反転し、井戸の上にグリースをリングの上に下に置く。 prにグリースにやさしく空気が溝に沿って井戸から脱出できることを押し下げイベントの空気の圧力の井戸で蓄積し、よく密封してください。ウェル内の空気の圧力上昇は、よくシール性を損なう、カバースライドを上げることができます。シッティングドロップ法では、光学的に透明な透明テープは井戸をカバーするために使用されます。したがって、このメソッドはシールですべての行/列に行われます。 </li><li>トレイが完了するまで次のウェルに移ります。 </li><li>セットアップ時にトレイを確認してください - ダスト粒子や他の汚染を排除するタンパク質結晶の偽陽性の識別を減らすことができます。 </li><li>あなたの実験を文書化するスコアリングシートを使用してください。 </li><li>一般的に4 ° Cと室温との間で、目的の培養温度でトレイを置きます。 20 ° Cは、最も一般的に成功しています。静かにトレイを処理し、どんな揺れを避けるために注意してください。インキュベーターの扉を開いたり閉じたりしながら注意してください。移転やインキュベーション中に、振動や温度変化は、結晶成長を防止または否定的に結晶品質に影響を与える可能性があります。物質を吸収するショック（例えばパッキンの泡）が結晶トレイの下に詰め物として使用することができます。 </li><li>結晶のトレイは、次の日を確認して、数日ごとに、常に注意してトレイを処理。トレイ特定のスコアリングシート内の任意の調査結果とドロップの形態を文書化します。結晶が時折はるかに早く（ほとんどすぐに）以降（最大で数ヶ月に！）表示されますが、結晶は、通常、表示に2-5日かかる。結晶化条件が特定されており、結晶の成長速度がわかれば、それは結晶が出現し、彼らの最終的な大きさの少なくとも半分に成長するまでトレイをそっとしておくのが最善の方法です。トレーが邪魔されないままにしておくと、それ故に大きな結晶の小さい数字を生成、結晶核生成のイベントの数を減らすことができます。 </li></ol> 2。手順をMicrobatch： <ol><li>タンパク質試料と沈殿剤の調製は、前述の通りである。 </li><li>エアスプレー、ほこりや他の大きな粒子を避けるために、新しいmicrobatchトレイ。 </li><li> microbatchトレイで、高3ミリメートル（井戸をカバーするだけの十分な）にパラフィン油をいっぱいに。油のアクセスを削除します。 </li><li>よく充填済み油にタンパク質溶液（50 mg / mLのリゾチーム）の1μLをロードする - 井戸の底（図2A）に直接ソリューションをピペッティングすることを確認してください。 </li><li>ドロップは、蛋白質の液滴で十分とヒューズの底に沈むことを確認 - よくトレイのセットアップ方式（図2B）によると中に沈殿剤溶液の1μLをロードする。 </li><li>トレイが完了するまで次のウェルに移動します。 </li><li>吊り下げ/シッティングドロップの手順のステップ11から14に従ってください。 </li></ol> 3。代表的な結果： 結晶化は通常、X線結晶構造解析のボトルネックと呼ば​​れています。沈殿条件のスパース行列不完全な階乗の画面は、通常、それらの大きな単結晶の中で、タンパク質の凝集と沈殿の多くの異なったタイプを生成します。タンパク質や沈殿剤濃度が高すぎる場合、1つは、明確な形と大きさ（アモルファス降水量）と茶色の問題を見ることができます。解決策が不飽和のされている場合、ドロップはしばしば降水量のあらゆる種類の完全に明確で欠いているだろう。図3は、降水現象と結晶（デッサウら 、2006）のためのいくつかの例を示しています。より詳細な解釈を持つより多くの降水現象は、次のURLで参照することができます<a href="http://xray.bmc.uu.se/terese/tutorials.html">http://xray.bmc.uu.se/terese/tutorials.html</a> 。 <img src="/files/ftp_upload/2285/2285fig1.jpg" alt="図1" /> 図1。タンパク質の結晶化の原理。 タンパク質の結晶化の原理。蒸気拡散実験（）で沈殿剤と蛋白質の等容量はドロップに存在しています。水が出て拡散すると平衡はドロップとリザーバー溶液との間で達成されるまで、沈殿剤やタンパク質濃度の両方が倍増されます。バッチ晶析では（B）沈殿剤と蛋白質の濃度は、実験中に変化しません。指して - タンパク質は不飽和のまま、結晶が、ポイントBを形成することができます - タンパク質の核生成が起こり、結晶が形成し始めたと溶液中のタンパク質の濃度は飽和状態に低下します。ポイントC - タンパク質は沈殿するが、結晶は依然として大​​きくなることがあります。キャピラリー差し込ま-ゲルを使用して（C）透析結晶化。塩は、ゲルのプラグを介して（および/または沈殿剤拡散タンパク質試料に）タンパク質試料から拡散するように結晶が現れます。 <img src="/files/ftp_upload/2285/2285fig2.jpg" alt="図2" /> 図2。典型的なタンパク質の結晶化実験の概要。 ハンギングドロップ蒸気拡散、シッティングドロップ蒸気拡散、およびmicrobatchタンパク質の結晶化のための（A）の手順。それぞれのケースでは、濃縮されたタンパク質試料の少量をprecipitanの等しいまたはより小さいボリュームと混合され、T溶液と平衡化させた。 （B）鶏リゾチーム、24ウェルトレーの結晶は塩化ナトリウムの様々な濃度（沈殿剤）とし、種々のpHにおいて緩衝剤として0.1 M酢酸ナトリウム（ - 4.9 4.0）と設定されています。 <img src="/files/ftp_upload/2285/2285fig3.jpg" alt="図3" /> 図3。タンパク質結晶化実験の典型的な成果。 （A）アモルファス沈殿。タンパク質や沈殿剤（または両方）を高濃度にある時。 （B）相分離。高濃度で一定の沈殿剤と混合したときにタンパク質や洗剤は、別のフェーズに分けることができます。 （C）AtCSN7のロッド状のタンパク質の結晶は、ポリエチレングリコール8000及び酢酸マグネシウム（デッサウら 、2006）で得られた。 （D）リゾチームの結晶は、1.0 M NaClと酢酸ナトリウムのpHを4.9に得られた。 （E）飽和滴の下で、通常はクリアのまま。モシェデッサウによる写真撮影。

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利害の衝突は宣言されません。

この記事では、タンパク質の結晶化のための一般的な現在のプロトコルを説明し、実証する。マルチステップの手順でそれ以来、いくつかの考慮事項は次のことに注意一つのニーズがあります。非常に少量（0.5-2μL）で作業する場合、蒸発による減少の乾燥は大きな懸念事項です。したがって、それは十分に制御された環境（低空気の流れと、高湿度と厳しい温度制御）で動作するように、?...

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		<tbody>
<tr>
<td>New Item</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<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>
</tr>
<tr>
<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>
<tr>
<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>
<td>&nbsp;</td>	</tr>	</tbody>
		</table>

protein crystallization for x-ray crystallography

彼らがどのように機能するか推測する生体高分子の三次元構造を使用すると、現代生物学の最も重要な分野の一つである。原子分解能構造の可用性は、タンパク質の機能の深さとユニークな理解を提供し、そして生きている細胞の内部の仕組みを解明するのに役立ちます。これまでに、蛋白質構造データバンク（RCSB - PDB）のエントリの86％がX線結晶学を用いて決定された巨大分子の構造です。 結晶解析に適した結晶を得るために、高分子（例えばタンパク質、核酸、タンパク質 - タンパク質複合体やタンパク質 - 核酸複合体）が均一にまで精製する必要がある、またはできるだけ近い均一になるまで。準備の均一性は高解像度（;マクファーソン、1999 Bergfors、1999）に回折する結晶を得るための重要な要素です。 結晶は、過飽和に高分子をもたらす必要があります。サンプルは、したがって、高分子の凝集または沈澱（通常は20〜50 mg / mL）を起こすことなく可能な限り高い濃度に濃縮する必要があります。沈殿剤にサンプルを導入することは、溶液から成長する大型の三次元結晶をもたらすことができます溶液中でのタンパク質結晶の核形成を促進することができます。蒸気拡散とバッチの結晶化：結晶を取得する2つの主なテクニックがあります。蒸気拡散、沈殿剤と蛋白溶液の混合物を含むドロップで純粋な沈殿剤とチャンバー内に封入されている。ドロップと沈殿剤の浸​​透圧は（図1A）等しくなるまで水蒸気は、ドロップから拡散する。均衡が達成されるまで滴の脱水は、理想的に相図の結晶核のゾーンで、タンパク質と沈殿剤の両方の遅い濃度を引き起こします。バッチ法は、沈殿剤の適切な量（図1B）とを混合タンパク質によって核のゾーンに直接タンパク質をもたらすに依存しています。このメソッドは、通常ドロップのうち水の拡散を防ぐために、パラフィン/鉱物油の混合物の下に実行されます。 ここでは、石油のもとでのバッチ結晶化に加えて、ドロップやシッティングドロップを吊り下げ、蒸気の拡散のための実験装置の二種類のデモンストレーションを行います。

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

分子の3次元構造がどのように分子の機能のユニークな理解を提供します。ニア原子分解能で構造決定のための主要な方法は、X線結晶です。ここで、我々はX線結晶構造決定に適している任意の高分子の三次元結晶を得るための現在の方法を示しています。

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 ...

protein-crystallization-for-x-ray-crystallography

Research

JoVE Journal

Biology

63.1K ビュー.  Yale University. 分子の3次元構造がどのように分子の機能のユニークな理解を提供します。ニア原子分解能で構造決定のための主要な方法は、X線結晶です。ここで、我々はX線結晶構造決定に適している任意の高分子の三次元結晶を得るための現在の方法を示しています。

ビデオ: 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.

シンクロトロンX線トモグラフィを用いたサブミクロン分解能を持つ非侵襲的3次元可視化

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 ...

生物学

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.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

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.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

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.

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 ...

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 ...

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.

前臨床X線コンピュータ断層撮影データセットの3Dプリント

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.

ランダムMicroseedマトリックススクリーニングによってタンパク質結晶化の成功率を向上させる

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.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</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.

効率的な哺乳動物細胞発現および結晶化のための細胞外糖タンパク質のシングルステップの精製

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