Name: microcrystallography of protein crystals and in cellulo diffraction
Uploaded: 2017-07-21T14:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

著者たちは、オーストラリアシンクロトロンのMX2ビームラインでの支援のために精製微結晶、Daniel ErikssonとTom Caradoc-Daviesの写真を提供するChan-Sien Layと、Monash UniversityのFlowCore施設からのKathryn FlanaganとAndrew Fryga貴重な援助。

<ol>
	<li>Tari, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+structural+biology+in+drug+discovery.">The utility of structural biology in drug discovery.</a> Methods Mol Bio (Clifton, N.J). 841, 1-27 (2012).</li><li>Gati, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+crystallography+on+in+vivo+grown+microcrystals+using+synchrotron+radiation.">Serial crystallography on in vivo grown microcrystals using synchrotron radiation.</a> IUCrJ. 1 (2), (2014).</li><li>Redecke, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natively+inhibited+Trypanosoma+brucei+cathepsin+B+structure+determined+by+using+an+X-ray+laser.">Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.</a> Science. 339 (2013), 227-230 (2013).</li><li>Shi, D., Nannenga, B. 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F., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-crystallography+comes+of+age.">Micro-crystallography comes of age.</a> Curr Opin Struct Biol. 22 (5), 602-612 (2012).</li><li>Boudes, M., Garriga, D., Coulibaly, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflections+on+the+Many+Facets+of+Protein+Microcrystallography.">Reflections on the Many Facets of Protein Microcrystallography.</a> Aust J Chem. 67 (12), 1793-1806 (2014).</li><li>Nelson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+cross-beta+spine+of+amyloid-like+fibrils.">Structure of the cross-beta spine of amyloid-like fibrils.</a> Nature. 435 (7043), 773-778 (2005).</li><li>Coulibaly, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+organization+of+cypovirus+polyhedra.">The molecular organization of cypovirus polyhedra.</a> Nature. 446 (7131), 97-101 (2007).</li><li>Coulibaly, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+atomic+structure+of+baculovirus+polyhedra+reveals+the+independent+emergence+of+infectious+crystals+in+DNA+and+RNA+viruses.">The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses.</a> Proc Natl Acad Sci U S A. 106 (52), 22205-22210 (2009).</li><li>Johansson, L. 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X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+crystallography+at+X-ray+free-electron+lasers:+the+next+generation+of+structural+biology.">In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology.</a> Phil Trans R Soc B. 369 (1647), 20130497 (2014).</li><li>Duszenko, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+protein+crystallization+in+combination+with+highly+brilliant+radiation+sources+offers+novel+opportunities+for+the+structural+analysis+of+post-translationally+modified+eukaryotic+proteins.">In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins.</a> Acta Cryst. F. 71 (8), 929-937 (2015).</li><li>Axford, D., Ji, X., Stuart, D. I., Sutton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+cellulo+structure+determination+of+a+novel+cypovirus+polyhedrin.">In cellulo structure determination of a novel cypovirus polyhedrin.</a> Acta Cryst D. 70 (5), 1435-1441 (2014).</li><li>Sawaya, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystal+structure+obtained+at+2.9+&#197;+resolution+from+injecting+bacterial+cells+into+an+X-ray+free-electron+laser+beam.">Protein crystal structure obtained at 2.9 &#197; resolution from injecting bacterial cells into an X-ray free-electron laser beam.</a> Proc Natl Acad Sci U S A. 111 (35), 12769-12774 (2014).</li><li>Tsutsui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Diffraction-Quality+Protein+Crystal+Processed+as+an+Autophagic+Cargo.">A Diffraction-Quality Protein Crystal Processed as an Autophagic Cargo.</a> Mol Cell. 58 (1), 186-193 (2015).</li><li>Boudes, M., Garriga, D., Fryga, A., Caradoc-Davies, T., Coulibaly, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pipeline+for+structure+determination+of+in+vivo-grown+crystals+using+in+cellulo+diffraction.">A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction.</a> Acta Cryst D. 72, 576-585 (2016).</li><li>Doye, J. P. K., Poon, W. C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystallization+in+vivo.">Protein crystallization in vivo.</a> Curr Opin Colloid Interface Sci. 11 (1), 40-46 (2006).</li><li>Mori, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Bombyx+mori+cytoplasmic+polyhedrosis+virus+polyhedrin+in+insect+cells+by+using+a+baculovirus+expression+vector,+and+its+assembly+into+polyhedra.">Expression of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin in insect cells by using a baculovirus expression vector, and its assembly into polyhedra.</a> J Gen Virol. 74, 99-102 (1993).</li><li>Arevalo, M. T., Wong, T. M., Ross, T. 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(76), (2013).</li><li>Otwinowski, Z., Minor, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+X-ray+diffraction+data+collected+in+oscillation+mode.">Processing of X-ray diffraction data collected in oscillation mode.</a> Methods Enzymol. 276, 307-326 (1997).</li><li>Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R., Leslie, A. G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iMOSFLM:+a+new+graphical+interface+for+diffraction-image+processing+with+MOSFLM.">iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM.</a> Acta Cryst D. 67, 271-281 (2011).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XDS.">XDS.</a> Acta Cryst D. 66, 125-132 (2010).</li><li>French, S., Wilson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+treatment+of+negative+intensity+observations.">On the treatment of negative intensity observations.</a> Acta Cryst.A. 34, 517-525 (1978).</li><li>Winn, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+CCP4+suite+and+current+developments.">Overview of the CCP4 suite and current developments.</a> Acta Cryst D. 67, 235-242 (2011).</li><li>Foadi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustering+procedures+for+the+optimal+selection+of+data+sets+from+multiple+crystals+in+macromolecular+crystallography.">Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography.</a> Acta Cryst D. 69 (8), 1617-1632 (2013).</li><li>Rey, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virology:+holed+up+in+a+natural+crystal.">Virology: holed up in a natural crystal.</a> Nature. 446 (7131), 35-37 (2007).</li><li>Sch&#246;nherr, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+investigation+of+dynamic+protein+crystallization+in+living+cells.">Real-time investigation of dynamic protein crystallization in living cells.</a> Struct Dyn. 2 (4), 041712 (2015).</li><li>Hasegawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+crystallization+of+human+IgG+in+the+endoplasmic+reticulum+of+engineered+Chinese+hamster+ovary+(CHO)+cells.">In vivo crystallization of human IgG in the endoplasmic reticulum of engineered Chinese hamster ovary (CHO) cells.</a> J Biol Chem. 286 (22), 19917-19931 (2011).</li><li>Ishchenko, A., Cherezov, V., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Delivery+of+Protein+Microcrystals+in+Lipidic+Cubic+Phase+for+Serial+Femtosecond+Crystallography.">Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography.</a> J Vis Exp. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+calcineurin+crystals+formed+using+the+baculovirus+expression+system.">In vivo calcineurin crystals formed using the baculovirus expression system.</a> Micros Res Tech. 34 (1), 77-86 (1996).</li><li>Nass, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Investigation of protein structure determination using X-ray free-electron lasers. , (2013).</li></ol>

著者は何も開示することはない。

このプロトコルは、過去に見過ごされていた非常に小さな結晶の分析を容易にする目的で、微結晶を分析する2つのアプローチを提供する。 微結晶の浄化のための重要なステップ 提示されたプロトコールは、モデル系としてSf9細胞で発現されたボンビクス・モリ CPV1ポリヘドリンを用いて最適化されている。しかしながら、 in vivo微結晶は?...

生物学的高分子の高分解能構造の決定のためのX線結晶学の使用は、過去20年間にわたり安定した進行を経験している。非専門家による研究者によるX線結晶学の益々の取り込みは、ライフサイエンスの多くの分野におけるこのアプローチの民主化を実証している1 。 歴史的に、約10μm以下の寸法を有する結晶は、構造決定のために使用できないとしても、困難であると考えられてきた。世界的なシンクロトロン放射源の専用マイクロフォーカスビームラインの利用可能性と、微結晶を操作するためのツールの開発などの技術的進歩により、X線マイクロ結晶の幅広い利用を妨げてきた多くの障壁が取り除かれました。シリアルX線microcrystallography 2,3及びマイクロ電子回折4ヘクタールの進歩構造決意用マイクロおよびナノ結晶の使用が可能でなく、大きな結晶5、6、7の使用に時々好ましくないだけであることが示されまし。 これらの進歩は、第一のペプチド8及び昆虫ウイルス9、10によって生成される天然の結晶の研究に適用しました。それらは現在、膜タンパク質や大きな複合体などの最も難しい系を含む多様な生物学的巨大分子に使用されています11 。これらの微結晶の分析を容易にするために、それらはメソ 、特に膜タンパク質12およびマイクロ流体チップ13において分析されている。 これらの新規な微結晶化方法の利用可能性は、古典的なインビトロ crystallogenesisに代わるものを提供する構造生物学14、15、16のための新たな経路として結晶体内。残念なことに、 インビボ結晶が産生されても、細胞からの精製中のリガンドの分解または喪失、シンクロトロンビームラインでの結晶の操作および視覚化の困難さ、退屈なX線回折実験などのいくつかの障害が残る。別の結晶はまた、任意の精製工程17、18、19せずに細胞内で直接分析されているように。比較分析はcelluloアプローチにおけるように精製された結晶およびより高い解像度20の収率データの分析よりも効率的であることを示唆しています。 このプロトコルはタンパク質微結晶学の新しい研究者を支援する傾向があった。それは、シンクロトロンビームラインでのX線回折実験のための試料調製および操作に焦点を当てた方法論を提供する。 2つの選択肢が、古典的な微結晶学のための単離された結晶またはフローサイトメトリーによってソーティングされた結晶含有細胞を用いて、セルロ分析で提案されている（ 図1 ）。

<table><tbody><tr><td>Sf9 cells</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>SF900-SFM insect medium</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>1 L cell culture flask</td><td>Thermofisher Scientific</td><td></td><td></td></tr><tr><td>Shaking incubator for insect cell culture</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>50 mL conical tubes</td><td>Falcon</td><td></td><td></td></tr><tr><td>Centrifuge with swing buckets for 50 mL tubes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Sonicator equiped with a 19 mm probe</td><td>MSE Soniprep 150&amp;nbsp;</td><td></td><td></td></tr><tr><td>Glass slides</td><td>Hampton Research</td><td></td><td></td></tr><tr><td>Hemacytometer</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Propidium iodide</td><td>Thermofisher Scientific</td><td></td><td></td></tr><tr><td>BD Influx cell sorter&amp;nbsp;</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Hampton Heavy atom screens</td><td>Hampton Research</td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Micromesh</td><td>Mitigen</td><td></td><td>700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td></tr><tr><td>Paper wick</td><td>Mitigen</td><td></td><td>The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used.</td></tr><tr><td>Ethylene glycol</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Trypan blue</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>MX2 microfocus beamline</td><td>Australian Synchrotron</td><td></td><td>A list of available microfocus beamlines can be found in Boudes &lt;em&gt;et al.&lt;/em&gt; (2014) &lt;em&gt;Reflections on the Many Facets of Protein&lt;br/&gt; Microcrystallography.&lt;/em&gt; Australian Journal of Chemistry 67 (12), 1793&amp;ndash;1806,&lt;br/&gt; doi:10.1071/CH14455.</td></tr></tbody></table>

microcrystallography of protein crystals and in cellulo diffraction

多くのシンクロトロン施設で高品質のマイクロフォーカスビームラインが出現したことにより、最大の次元で10μm以下の結晶の日常的な解析が可能になりました。本発明者らは、X線結晶学によるタンパク質微結晶の構造決定のための2つの代替のワークフローを提示し、特にin vivoで成長した結晶に焦点を当てる。微結晶は、超音波処理によって細胞から抽出され、微分遠心分離によって精製されるか、または結晶含有細胞のフローサイトメトリーによる細胞選別の後、セルロで分析される。場合によっては、精製された結晶または結晶含有セルを、実験段階的な調整のために重原子溶液に浸漬する。これらの試料は、マイクロメッシュ支持体上に塗布し、液体窒素中でフラッシュ冷却することにより同様の方法で回折実験用に調製する。我々は、単離された微結晶および結晶質結晶の連続回折実験を簡単に説明し、モデル構築および精密化に適したデータセットを生成するためにマイクロフォーカスシンクロトロンビームラインを使用している。 これらのワークフローは、昆虫細胞に組換えバキュロウイルスを感染させることによって産生されるBombyx mori cypovirus 1（BmCPV1）ポリヘドリンの結晶で例示される。このケーススタディでは、セルロ分析では精製された結晶の分析よりも効率的であり、発現から精製まで約8日間で構造が得られます。

生体内微結晶を用いた構造決定のための代替方法の両方の概要が示されている（ 図1 ）。多面体は、超音波処理および遠心分離によって容易に精製することができる。密度のため、ピペットで取り除くことができる破片の層の下のチューブの底に層を形成する（ 図3aおよび3b ）。次いで、サンプ?...

Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

Microcrystallography of Protein Crystals and In Cellulo Diffraction

タンパク質微結晶を用いたX線結晶解析のためのプロトコールが提示されている。精製後又はcelluloにおける微結晶-grown インビボで分析する2つの例が比較されます。

タンパク質結晶の微結晶化および<em&gt;セルロで</em&gt;回折

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

microcrystallography-of-protein-crystals-and-in-cellulo-diffraction

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

9.0K ビュー.  Monash University. タンパク質微結晶を用いたX線結晶解析のためのプロトコールが提示されている。精製後又はcelluloにおける微結晶-grown インビボで分析する2つの例が比較されます。 

ビデオ: Microcrystallography of Protein Crystals and In Cellulo Diffraction

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

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

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

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

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

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 ...

化学

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 ...

Microcrystal Electron Diffraction of Small Molecules

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.

Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments.

低分子の微小結晶電子回折

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been ...

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 ...

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 ...

生物学

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 ...

High-Throughput Protein Crystallization via Microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature. This protocol describes a straightforward high-throughput (HTP) workflow for screening crystallization conditions through the use of a 96-well dialysis plate. These plates follow the Society for Biomolecular Screening (SBS) standard and can be easily set up using any standard crystallization laboratory. Once optimal conditions are identified, large quantities of crystals (hundreds of microcrystals) can be produced using the dialyzer. To validate the robustness and versatility of this approach, four different proteins were crystallized, including two membrane proteins.

High-Throughput Protein Crystallization via Microdialysis

The presented protocol describes a straightforward approach for screening protein crystallization conditions and crystal growth using a 96-well high-throughput dialysis plate. The use of dialyzer tubes for the large-scale growth of microcrystals is also demonstrated for serial crystallography and MicroED applications.

マイクロダイアリシス による ハイスループットタンパク質結晶化

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug ...

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 ...

Protein Crystallization

Protein crystallization, obtaining a solid lattice of biomolecules, elucidates protein structure and enables the study of protein function. Crystallization involves drying purified protein under a combination of many factors, including pH, temperature, ionic strength, and protein concentration. Once crystals are obtained, the protein structure can be elucidated by x-ray diffraction and computation of an electron density model.
This video introduces protein crystallization and shows a general procedure. Protein expression and purification, crystallization, and x-ray diffraction are covered in the procedure. Applications of protein crystallization include in silico drug design, binding site determination, and membrane protein structure analysis.
<div class="chapter_text" id="script_0">
Protein crystallization is the process of obtaining a latticed solid form of a protein. These crystals are especially valuable to structural biologists, assisting in the study of protein function. Other techniques, such as mass spec or SDS-PAGE, can only provide information on the one-dimensional structure of proteins. Protein crystallization is complemented by the techniques of recombinant protein expression and x-ray diffraction. This video will show the principles of protein crystallization, a general laboratory procedure, and several of its applications in the biochemical field.
</div>
<div class="chapter_text" id="script_1">
The first step required in the process is to obtain milligram quantities of very pure protein, typically using recombinant protein expression. The gene corresponding to the protein of interest is cloned into an expression vector, and the expressed protein is fused to an affinity tag, such as poly-histidine, to assist in the purification by affinity chromatography. To learn more, see this collection&#39;s video on affinity chromatography.
Formation of the purified protein into crystals is dependent on the proper combination of many factors, including pH, ionic strength, concentrations of precipitant and protein, temperature, and rate of equilibration. The most common method used is vapor diffusion, of which there are two categories: hanging drop and sitting drop. A droplet containing pure protein, buffer, and precipitant, which is an ionic solid that binds water molecules, reducing water availability for the protein and mimicking higher protein concentration, is in an enclosed microwell with a reservoir with a more highly concentrated mixture of the same buffer and precipitant. At the beginning, the concentrations of protein and precipitant are too low to cause crystallization. During the course of the experiment, water vaporizes from the droplet and collects in the reservoir; a decrease in the amount of water in the droplet causes the system to become supersaturated, and nucleation, followed by crystallization, can occur. The net transfer of water from the droplet is in equilibrium, and the system is maintained until the process is complete.
To visualize the 3D structure, x-ray diffraction is used. To obtain x-ray data from a crystal, it is placed in a monochromatic x-ray beam, where it is exposed to the beam at all angles. Each exposure provides an image, where each spot is a diffracted x-ray, which emerges from the crystal and is registered by a detector. The data are combined to produce a model of the arrangement of atoms within the crystal. The resulting crystal structure demonstrates the 3-dimensional placement of the atoms, with a typical resolution of 2 angstroms.

</div>
<div class="chapter_text" id="script_2">
Now that we have covered the principles of protein crystallization, let us look at a generalized protocol.
To begin the procedure an expression vector containing the gene of interest is transformed into cells. The cells are incubated and at mid-log phase, expression is initiated by adding an inducer, such as IPTG, which triggers transcription of the gene&#39;s mRNA. After protein expression, the crude material is suspended in lysis buffer, and then clarified by centrifugation.
The clarified lysate is then loaded onto a nickel column, and the polyhistidine-tagged protein binds to the column while all other biomolecules are washed away.
Once several milligrams of pure protein have been obtained, it is ready for crystallization by vapor diffusion. A 24-well hanging/sitting drop tray is filled with varying concentrations of sodium chloride and sodium acetate buffer solutions. For the sitting drop method, equal volumes of protein and reservoir solution are pipetted onto the shelf above each well, and then the tray is covered with transparent tape. The tray is then placed in an incubation chamber, and the wells are monitored for growth the following day, then every few days.
Once a proper crystal has been obtained it is ready for x-ray diffraction analysis. The crystal is mounted on a goniometer to position the crystal at selected orientations. The crystal is illuminated with a monochromatic beam of x-rays at all angles, producing a diffraction pattern. The software converts the two-dimensional images, taken at different orientations, to a three-dimensional model of the density of electrons within the crystal by determining the positions of the atoms in the crystal.
</div>
<div class="chapter_text" id="script_3">
Now that we have reviewed a procedure, let&#39;s review some useful applications of protein crystallization, and another crystallization technique.
Protein crystallization may be used for in silico drug design. The three-dimensional structure of Influenza virus&#39;s polymerase basic protein 2, which has been linked to viral infection in mammals, was determined by crystallization and x-ray diffraction. Potential binding sites in the protein are visualized, and with the use of a docking program, a three-dimensional molecule was designed that would insert into a cleft in the protein.
Co-crystallization of protein-DNA complexes is also a useful technique. DNA-binding proteins modulate a wide variety of biological functions such as transcription and DNA polymerization and DNA repair; and crystal structures of these complexes can provide insight into protein function, mechanism, and the nature of the specific interaction. The E. coli protein SeqA, a negative regulator of DNA replication, was co-crystallized with hemimethylated DNA.
Integral membrane proteins such as G-protein coupled receptors, or GCPRs, are difficult to crystallize due to their limited amount of polar surface area available for forming crystal lattice contacts, which has led to the development of fusion-protein-assisted protein crystallization. Genes encoding &#946;2 adrenergic receptor, a GCPR, and a lysozyme were inserted into an expression vector. The crystallization of the &#946;2AR- lysozyme fusion protein was achieved due to the increased extracellular hydrophilic surface over the naturally hydrophobic &#946;2AR, provided by the lysozyme, necessary for forming packing interactions in the crystal lattice.
</div>
<div class="chapter_text" id="script_4">
You&#39;ve just watched JoVE&#39;s video on protein crystallization. This video described its principles, a generalized protocol, and some its uses in the biomedical field. Thanks for watching!
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タンパク質結晶の微結晶化および

要約

さらに動画を探す

この動画の章

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

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

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

低分子の微小結晶電子回折

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

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

室温でのオンチップ結晶化と大規模シリアル回折

マイクロダイアリシスによるハイスループットタンパク質結晶化

X -線結晶構造のためのタンパク質の結晶化

タンパク質の結晶化

タンパク質結晶の微結晶化および

要約

さらに動画を探す

この動画の章

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

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

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

低分子の微小結晶電子回折

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

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

室温でのオンチップ結晶化と大規模シリアル回折

マイクロダイアリシス による ハイスループットタンパク質結晶化

X -線結晶構造のためのタンパク質の結晶化

タンパク質の結晶化

マイクロダイアリシスによるハイスループットタンパク質結晶化