Name: microfluidic chips for in situ crystal x-ray diffraction and in situ dynamic light scattering for serial crystallography
Uploaded: 2018-04-24T13:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography at JoVE.com

この作品は、ピア シード基金 PIF-2015-46 によって支えられた、BMBF 付与、ドイツの '超高速イメージング-構造、ダイナミクス、原子スケールでの物質の制御のためのハンブルク中心部' 卓越したクラスターと 05K12GU3、05K16GUA研究振興協会 (DFG)。自由電子レーザー研究センターと共著の作業指向プログラム資金によるヘルムホルツ協会によって資金を供給されました。EMBL ハンブルクでペトラ III ストレージ リング (DESY, ハンブルク, ドイツ) で放射光 MX データを集めた P14 運営のビームラインが。

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窓材として材料とポリイミド箔を充填エポキシ樹脂のパターニングによる x 線回折その場観察のためのマイクロ流体デバイスを作製します。私たちの手順は、以前 x 線チップ デザイン16,21以上作製プロセスの様々 なステップを最適化されています。我々 はウィンドウの厚さを減少、それにより作製も緩和しながら散乱背景として少ないプロセスの手順が必要です。その場で結晶化が記述されているプロトコルを使用してには、実質的な利点があります。室温で回折データ収集し、それによっていくつかのケースでタンパク質構造の人工物を導入するリスクを含んだ低温保護の必要性を除外します。さらに、ネイティブな環境から結晶の転送を避けることができるため、結晶はありません物理的なストレスの対象となります。このプロシージャを介して結晶は最高品質を維持し、任意の治療に苦しむしません。
私たちの経験ではプロトコルの中で最も重要な手順の結晶化プロセスを制御する中心です。適切な寸法と適切な結晶を x 線を取得するパラメーターを経験的に識別する必要があり、蒸気拡散実験から直接撮影することはできません。同一濃度のタンパク質と鉛塩法を使用して常にされませんでした別のチップ、または時同じチップ内でさまざまな坑井の結晶。これは結晶の核生成と成長に影響を与えるすべての要因を (蒸発軌道) を通じて母酒の組成や結晶化速度など、慎重に検討する必要があることを示します。大きい水晶は、高い解像度に回折、適当に大きな結晶が理想的に成長してください。結晶核生成と成長のプロセスは、DLS 測定後すること。~ 50 μ m の中レーザーの焦点を調整するチップの薄い結晶コンパートメントは挑戦することができ、慎重に手動位置合わせを必要とする場合があります。100 μ m よりも深い井戸を用いたレーザー自動整列だった可能で信頼性が高く、自動取得スキームを通じて複数の井戸を監視することが。
ポリイミド ベース x 線チップ生産のみ低バック グラウンドと 3 つのタンパク質のモデル構造を解くことによってルーチン x 線回折データ収集のためのこれらの装置の適合性を示します。大きくタンパク質結晶と従来の x 線データ コレクションから以前達成の解像度と比較してチップの最高解像度が異なっていた。さらに結晶化条件の最適化がさらに回折を向上、これはいくつかの要因かもしれません。最大適用クリスタルが 30 μ m より小さい寸法 1.8 Å 分解能その場回折データを収集することが可能だった。タウマチン回折データの詳細な分析は、放射線被害についての洞察を提供しました。放射線被害の拡張を制限するには、隣接結晶とラジカルの拡散が起こり、マイクロ流体デバイスのコンパートメントあたり 1 つだけの単結晶を公開する必要があります。データ収集の速度を向上させる、これを将来に自動化する必要があります。
結晶形態のためいくつかのケースで配向が発生ことができます。これだったたとえばチオレドキシン データセットの場合、結晶強く配向チップ windows を基準。ここでも、完全な回折データセットを収集することができます。結晶がチップの配向を展示し、特に対応する空間群にも低対称性がある場合、データセットの完全性監視してくださいコレクションされている十分な回折パターン杖、その場合収集。
ポンプ ・ プローブによる反応を誘起光を用いた場合、これらのチップを用いた時間分解研究が直接可能です。ポンプ レーザー用ポリイミド箔の光透過が解明する、また、ポリイミドを光学的にオフする必要があります。 または coc 認証も使用できます。現在のマイクロ形状は基板結晶が成長した後に実験を混合できないようにします。しかし、このような両方の時間分解 x 線回折のためのデザインを混合し同様散乱アプローチ19にも適応するのに記載されている x 線チップ作製プロトコルを見込んでいます。

蛋白質の 3 D 構造を知ることは、その機能を理解することが不可欠です。近く原子分解能の構造は、最もよく、x 線結晶構造解析によって得られるところ。このテクニックを公開タンパク質結晶 x 線放射と精製と構造決定の結果として得られる回折パターンを分析しています。伝統的な x 線結晶構造解析、完全な回折データセットは、極低温における単一、理想的には大きなクリスタルから記録されます。ただし、このような結晶が成長し、些細な大抵ないと適当な低温保存条件を識別することができますそれ自体困難になるし、時々 またネイティブ蛋白質構造5からの逸脱を引き起こす可能性があります。
X 線自由電子レーザ (FEL) および放射光ビームラインにおける最近の技術の進歩より小さい結晶構造を解決するために許可している、新しいマイクロ集束ビームライン増加 x 線ビームの輝きと改善された x 線検出器になった利用可能な6,7。通常、小さい水晶はより大きく成長し、欠陥の遊離結晶8,9やすくなっています。しかし、小さな結晶 x 線照射損傷の大きい水晶よりもはるかに高速に苦しみます。これは大きな結晶に比較するため、同等の解像に回折により小さい結晶ボリュームに高い x 線の線量を投影する必要があります。したがって、でも低温保護はしばしば不十分である単一微小結晶から完全な回折データ セットを記録です。
このハードルを克服するためにシリアルの結晶を回収して完全なデータセットを取得する多くのランダム配向結晶からの回折パターンを結合する選択の方法となっています。放射線 x 線線量が合計を広めることで誘起結晶被害を最小限高結晶5,10数以上のタンパク質の構造を解決するために使用します。'回折破壊する前に' 自由電子レーザー実験、各結晶はあくまでフェムト秒 x 線パルスを使用して 1 つの露出のため。第 3 世代放射光源でマイクロ フォーカス ビームライン順番数ミリ秒短い x 線露光11,12,13,14シリアル結晶構造解析を実行できます。水晶発振またはデータ収集中に回転、しかし、部分的なブラッグ反射だけ記録できる、それゆえ数万人またはより多くの回折パターンが通常構造決定15必要。これまでサンプル配信方法の多様なセットとして開発されたシリアルの結晶学の最近の見直し14,16,17,18,19。中で、いくつかの固定ターゲット ベース サンプル配信など大幅より少ない回折パターンを提供できる、同様に完全なデータセットも低消費しながら正常に x 線露光中に結晶回転と結合された戦略サンプルと比較して古典的なシリアルの結晶学への静止画が、実験記録7,16,20,21,22,23,24。
低 x 線バック グラウンドを持つマイクロ流体デバイスを作製するためのプロトコルを提案します。デバイスのソフト ・ リソグラフィーを用いた 5 分エポキシ接着剤からパターン化されているし、部屋の温度でその場x 線回折実験の場合として x 線セットアップに直接試料を統合するから利益を得るために適しています。次のミキシング誘起動力学18,19時間分解の研究。マイクロ流路、高分子ポリイミド箔、複合厚低 x 線背景イメージングを可能にする約 16 μ m で x 線窓の両側に蓋をします。すべての使用された材料は、良い耐溶剤性を提供します。この工法は、比較的簡単で安価なです。SU 8 マスター基板の調達後、典型的な研究ラボ設定でクリーン ルーム外すべての製造を完了できます。
ゴニオ メーター ベースの固定ターゲット シリアル結晶構造解析アプリケーションの例では、チップをについて説明します。まず、microfluidically ナノリットル サイズの液滴の選択した番号に水性反応を分割するキャピラリー valving を使用しての設計と試作に関する考慮事項を説明します。このローディング機構はチャネルのデッド ボリュームからサンプル損失を回避し、分割することができます簡単に手動で実行する流体の作動のためのポンプやその他の機器を使用せず。タンパク質溶液のような分離ナノリットル サイズ滴監視対象はその場で動的光散乱 (DLS) を使用して制御タンパク質の結晶核生成と成長します。以前、DL 測定をガラス スライド25,26に結合したポリジメチルシロキサン (PDMS) 構造体から成るマイクロ流体デバイスで実行できることが実証されています。ポリイミド膜は 550 より波長が長いため高伝送のため nm、アプローチを拡張する単位の x 線透明チップだけでなく、適切なレーザー波長27,28を使用する場合。DL の結果に基づいて、初期核生成が観察できますとさらに液滴蒸発を停止して、少ないが大きなタンパク質の結晶を得ることができます。
十分な結晶が成長した、完全な x 線回折データセット、収集できます室温でターゲット シリアル x 線回折その場固定ベース角度計を使用します。回折データセットは、蛋白質結晶構造を解決するために一連のソフトウェア ツールとカスタム スクリプトを使用して処理されます。この手法は、従来の結晶学実験で使用される低温保存中に生じる工芸品を回避できます。
私たちは約を使用して解決された 3 つの蛋白質ターゲット構造比較 10-20 μ m の小さな結晶が良いし、2 Å 分解能にチップで成長して。結晶化とその場で、処理およびそれ故に機械的回折によって壊れやすい結晶の妨害が最小限に抑えられます。このプロトコルは、タンパク質結晶回折の高解像度と低解像度 (3.0 Å に 1.7 Å) に適用できます。ほぼすべてのクリスタルは回折のために使えるので、小さなサンプルは、非常に効率的な結晶配信メソッドに浪費されています。
このプロトコルは、蛋白質結晶化と回折データ収集その場x 線透明マイクロ流体チップを準備する方法の詳細なガイドを提供します。手順は、実験室での高度な機器を必要とせずマイクロ精度の恩恵を慎重に設計されました。また、非専門家による専門的なゴニオ メーターや、結果の再現を容易にするため加湿器を必要とせず放射光ビームラインでのデータ コレクションを実行できます。紹介したテクニックは、低温保護または結晶処理によるシリアル ミリ秒結晶学データ収集のため室温照射損傷を最小限に維持しながら、成長後の結晶に応力を導入することがなく適用できます。したがって、この方法はタンパク質結晶化プロジェクトに適しています。

<table><tbody><tr><td>SU-8 3000 Series</td><td>MicroChem Corp.</td><td>SU-8 3000</td><td>Photoresist</td></tr><tr><td>PGMEA</td><td>Sigma-Aldrich</td><td>484431</td><td>Developer</td></tr><tr><td>Isopropyl alcohol</td><td></td><td></td><td>Solvent</td></tr><tr><td>Ethanol</td><td></td><td></td><td>Solvent</td></tr><tr><td>Epoxy glue</td><td>UHU</td><td>Plus Schnellfest 5 min</td><td>Epoxy glue</td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184</td><td>Silicone</td></tr><tr><td>Kapton foil</td><td>Dupont/ American Durafilm</td><td>HN grade, gauge 30 (7.5 &amp;mu;m)</td><td>polyimide foil</td></tr><tr><td>APTS</td><td>Sigma-Aldrich</td><td>440140</td><td>Chemical</td></tr><tr><td>GPTS</td><td>Sigma-Aldrich</td><td>440167</td><td>Chemical</td></tr><tr><td>Cytop CTX-109AE</td><td>Asahi Glass Co. Ltd</td><td>Cytop CTX-109AE</td><td>Cytop fluoropolymer coating</td></tr><tr><td>CT-Solv 100E</td><td>Asahi Glass Co. Ltd</td><td>CT-Solv 100E</td><td>Cytop fluoro-solvent</td></tr><tr><td>HFE-7500</td><td>3M</td><td>Novec 7500</td><td>Fluorinated oil</td></tr><tr><td>AutoCAD</td><td>AutoDesk Inc.</td><td>AutoCAD</td><td>CAD Software</td></tr><tr><td>Biopsy Punch</td><td>Harris</td><td>Uni-core 0.75 mm</td><td></td></tr><tr><td>Photo mask</td><td>JD Photo Data</td><td></td><td></td></tr><tr><td>3 inch wafer</td><td>University Wafer</td><td></td><td>Silicon wafer</td></tr><tr><td>Mask aligner</td><td>S&amp;Uuml;SS MicroTec</td><td>MJB4</td><td>Mask aligner</td></tr><tr><td>PDMS mixer</td><td>Thinky</td><td>ARE-250</td><td></td></tr><tr><td>Plasma machine</td><td>Diener electronic</td><td>Zepto</td><td></td></tr><tr><td>Thaumatin</td><td>Sigma Aldrich</td><td>T7638</td><td>Protein</td></tr><tr><td>Glucose Isomerase</td><td>Hamton Research</td><td>HR7-102</td><td>Protein</td></tr><tr><td>Bis-Tris</td><td>Sigma Aldrich</td><td>B9754</td><td>Chemical</td></tr><tr><td>Sodium Tartrate</td><td>Merck</td><td>106664</td><td>Chemical</td></tr><tr><td>Tris-HCl</td><td>Sigma Aldrich</td><td>10812846001</td><td>Chemical</td></tr><tr><td>HEPES</td><td>Carl Roth</td><td>6763.2</td><td>Chemical</td></tr><tr><td>Magnesium Chloride</td><td>Sigma Aldrich</td><td>208337</td><td>Chemical</td></tr><tr><td>Ammonium Sulfate</td><td>Sigma Aldrich</td><td>A4418</td><td>Chemical</td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E6758</td><td>Chemical</td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>1064060250</td><td>Chemical</td></tr><tr><td>PEG1500</td><td>Molecular Dimensions</td><td>MD2-100-6</td><td>Chemical</td></tr><tr><td>SPG buffer</td><td>Jena Bioscience</td><td>CSS-389</td><td>Chemical</td></tr><tr><td>SpectroLight600</td><td>XtalConcepts</td><td></td><td>DLS Instrument</td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td></td><td>Spectrophotometer</td></tr><tr><td>Zentrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Ultimaker2</td><td>Ultimaker</td><td></td><td>3D printer</td></tr><tr><td>Form2</td><td>Formlabs</td><td></td><td>3D printer</td></tr><tr><td>Amicon Filter</td><td>Sartorius Stedim</td><td></td><td>0.2 &amp;micro;m filter</td></tr><tr><td>Tubing</td><td>Adtech Polymer Engineering Ltd</td><td>Bioblock/05</td><td>PTFE tubing 0.3 mm Inner Diameter x 0.76 mm Outer Diameter</td></tr><tr><td>Syringes&amp;nbsp;</td><td>BD</td><td>309628</td><td>1ml Luer-Lock Tip</td></tr><tr><td>Needle&amp;nbsp;</td><td>Terumo Agani Needle</td><td>AN*2716R1</td><td>27Gx5/8&quot;</td></tr></tbody></table>

microfluidic chips for in situ crystal x-ray diffraction and in situ dynamic light scattering for serial crystallography

このプロトコルでは、ゴニオメータ ベースの固定ターゲット シリアル結晶構造解析のために最適化された低 x 線バック グラウンドで加工マイクロ流体デバイスについて説明します。デバイスはソフト ・ リソグラフィーを用いたエポキシ接着剤からパターン化されているし、室温でその場x 線回折実験に適しています。サンプル井戸低 x 線バック グラウンドを持つ回折データ収集を許可する高分子ポリイミド箔窓の両側には、蓋をします。この製作方法は多くを求めない安価です。SU 8 マスター基板の調達後、典型的な研究ラボ環境でクリーン ルーム外すべての製造を完了できます。チップの設計と試作のプロトコルは、microfluidically 定義ナノリットル サイズの液滴に水性反応を分割するキャピラリー valving を利用します。このローディング機構チャネル デッド ボリュームからサンプル損失を回避し、簡単に流体の作動のためのポンプやその他の機器を使用せず手動で実行することができます。我々 はどのように分離ナノリットル サイズ タンパク質溶液滴できますが監視対象の in situ動的光散乱制御タンパク質の結晶核生成と成長にについて説明します。適切な結晶を成長した後、室温でターゲット シリアル x 線回折その場固定ベース ゴニオ メーターを使用して完全な x 線回折データセットを収集できます。プロトコルは、ソフトウェア ツールのスイートを使用して解決し、蛋白質結晶構造を絞り込む回折データセットを処理するカスタム スクリプトを提供します。このアプローチでは、工芸品の低温保存または従来の結晶学実験の処理マニュアルの結晶中に誘発する可能性を回避できます。チップで育つ約 10-20 μ m の寸法で小さい水晶を使用して解決された 3 つの蛋白質の構造を比較し、.結晶化とその場で、処理およびそれ故に機械的回折によって壊れやすい結晶の妨害が最小限に抑えられます。プロトコルは、その場でシリアル結晶構造解析に適したカスタム x 線透明マイクロ流体チップを作製する方法を説明します。ほぼすべてのクリスタルは、回折データ収集の使用ことができます、これらのマイクロ チップ、非常に効率的な結晶配信方法です。

Watch this Scientific Journal Video about Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography at JoVE.com

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

このプロトコルでは、作製し、室温での x 線回折データ収集のためのマイクロ流体デバイスを操作する方法について詳しく説明します。さらに、動的光散乱法によるタンパク質の結晶化を監視する方法を説明し、処理し、分析する方法得られた回折データ。

シリアル結晶の結晶 x 線回折その場その場での動的光散乱のためのマイクロ流体チップ

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

microfluidic-chips-for-situ-crystal-x-ray-diffraction-situ-dynamic

Research

JoVE Journal

Chemistry

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

14.6K ビュー.  DESY. このプロトコルでは、作製し、室温での x 線回折データ収集のためのマイクロ流体デバイスを操作する方法について詳しく説明します。さらに、動的光散乱法によるタンパク質の結晶化を監視する方法を説明し、処理し、分析する方法得られた回折データ。

ビデオ: Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

化学固定することによりX線蛍光イメージングのための接着細胞の準備

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

化学

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

タンパク質結晶の成長を使用して異なる寸法自動結晶化その場の動的光散乱と相まって

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

生化学

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

保存科学における粉末X線回折：遺産アートオブジェクトに対する腐食製品のルーチンの結晶構造の決意に向けて

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

High Pressure Single Crystal Diffraction at PX^2

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the GSECARS 13-BM-C beamline at the Advanced Photon Source. The DAC program at 13-BM-C is part of the Partnership for Extreme Xtallography (PX^2) project. BX-90 type DACs with conical-type diamond anvils and backing plates are recommended for these experiments. The sample chamber should be loaded with noble gas to maintain a hydrostatic pressure environment. The sample is aligned to the rotation center of the diffraction goniometer. The MARCCD area detector is calibrated with a powder diffraction pattern from LaB6. The sample diffraction peaks are analyzed with the ATREX software program, and are then indexed with the RSV software program. RSV is used to refine the UB matrix of the single crystal, and with this information and the peak prediction function, more diffraction peaks can be located. Representative single crystal diffraction data from an omphacite (Ca0.51Na0.48)(Mg0.44Al0.44Fe2+0.14Fe3+0.02)Si2O6 sample were collected. Analysis of the data gave a monoclinic lattice with P2/n space group at 0.35 GPa, and the lattice parameters were found to be: a = 9.496 &#177;0.006 &#197;, b = 8.761 &#177;0.004 &#197;, c = 5.248 &#177;0.001 &#197;, &#946; = 105.06 &#177;0.03&#186;, &#945; = &#947; = 90&#186;.

In this report, we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell at the GSECARS 13-BM-C beamline at the Advanced Photon Source. ATREX and RSV programs are used to analyze the data.

PX ^ 2の高圧単結晶回折

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

背景散乱を最小限に抑えた高分子X線結晶学用オールインワンサンプルホルダー

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

ICPMSサンプル導入のためのマイクロ流体チップ

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

生物工学

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder microdiffraction two-dimensional (2D) maps at beamline 12.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Measurements can be performed on any sample that is less than 10 cm x 10 cm x 5 cm, with a flat exposed surface. The experimental geometry is calibrated using standard materials (elemental standards for XRF, and crystalline samples such as Si, quartz, or Al2O3 for diffraction). Samples are aligned to the focal point of the x-ray microbeam, and raster scans are performed, where each pixel of a map corresponds to one measurement, e.g., one XRF spectrum or one diffraction pattern. The data are then processed using the in-house developed software XMAS, which outputs text files, where each row corresponds to a pixel position. Representative data from moissanite and an olive snail shell are presented to demonstrate data quality, collection, and analysis strategies.

We describe a beamline setup meant to carry out rapid two-dimensional x-ray fluorescence and x-ray microdiffraction mapping of single crystal or powder samples using either Laue (polychromatic radiation) or powder (monochromatic radiation) diffraction. The resulting maps give information about strain, orientation, phase distribution, and plastic deformation.

・鉱物及び岩石試料の蛍光を放射光 x 線回折

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder ...

工学

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

酵素の結晶化と構造決定:多用途マイクロ流体チップにおける連続結晶学による基質複合体

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

In Situ X線回折研究のためのマイクロダイアルシスによるチップ上のタンパク質の結晶化

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

室温でのオンチップ結晶化と大規模シリアル回折

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

シリアル結晶の結晶 x 線回折その場その場での動的光散乱のためのマイクロ流体チップ

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