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

Forfatterne vil gjerne anerkjenne Chan-Sien Lay for å gi bilder av rensede mikrokrystaller, Daniel Eriksson og Tom Caradoc-Davies for støtte på MX2-strålen i den australske synkrotronen, og Kathryn Flanagan og Andrew Fryga fra FlowCore-anlegget ved Monash University for deres Uvurderlig hjelp.

Merk: In vivo krystallisering har blitt rapportert i mange organismer, inkludert i bakterier, gjær, planter, insekter og pattedyr (omtalt i referanse 21 ). Krystallisering av rekombinante proteiner har også blitt oppnådd i laboratoriet ved bruk av forbigående transfeksjon av pattedyrceller og baculovirusinfeksjon av insektceller. Følgende protokoll er blitt utviklet ved bruk av Bombyx mori cypovirus 1 (BmCPV1) polyhedrin-genet klonet i et rekombinant baculovirus under bacul...

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Denne protokollen gir to tilnærminger til å analysere mikrokrystaller med sikte på å lette analysen av svært små krystaller som tidligere hadde blitt oversett. Kritiske skritt for mikrokrystallrensing Den presenterte protokollen er optimalisert ved bruk av Bombyx mori CPV1 polyhedrin uttrykt i Sf9-celler som modellsystem. Imidlertid viser in vivo mikrokrystaller en stor variasjon i mekanisk motstand. For eksempel er cathepsin B nålignende krys...

Bruken av røntgenkrystallografi for bestemmelse av høyoppløsningsstrukturer av biologiske makromolekyler har opplevd en jevn progresjon de siste to tiårene. Den voksende opptaket av røntgenkrystallografi av ikke-ekspertforskere eksemplifiserer demokratisering av denne tilnærmingen på mange områder av biovitenskap 1 . Historisk sett har krystaller med dimensjoner under ~ 10 μm blitt betraktet som utfordrende, om ikke ubrukelige, for strukturbestemmelse. Den økende tilgjengeligheten av dedikerte mikrofokus stråler i synkrotron strålingskilder over hele verden og teknologiske fremskritt, for eksempel utviklingen av verktøy for å manipulere mikrokrystaller, har fjernet mye av disse barrierer som stymied den brede bruken av røntgen mikrokrystallografi. Fremskritt i seriell røntgenmikrokrystallografi 2 , 3 og mikroelektrondiffraksjon 4 haHar vist at bruken av mikro- og nanokrystaller for strukturbestemmelse ikke bare er mulig, men også noen ganger foretrukket for bruk av store krystaller 5 , 6 , 7 . Disse fremskrittene ble først anvendt på studiet av peptider 8 og naturlige krystaller produsert av insektvirusene 9 , 10 . De brukes nå til et mangfoldig utvalg av biologiske makromolekyler, inkludert de vanskeligste systemene som membranproteiner og store komplekser 11 . For å lette analysen av disse mikrokrystaller, har de blitt analysert i meso , spesielt membranproteiner 12 og i mikrofluidikk-chips 13 . Tilgjengeligheten av disse nye mikrokrystallografimetodikkene har økt muligheten for å bruke iVivo-krystallisering som en ny rute for strukturell biologi 14 , 15 , 16 som tilbyr et alternativ til klassisk in vitro- krystallogenese. Dessverre, selv når in vivo krystaller kan fremstilles, forblir flere hindringer som for eksempel nedbrytning eller tap av ligander under rensning fra celler, vanskeligheter med manipulering og visualisering av krystallene ved synkrotronstrenglinjen og kjedelige røntgendiffraksjonseksperimenter. Som en alternativ krystaller har også blitt analysert direkte i cellen uten noe rensingstrinn 17 , 18 , 19 . En komparativ analyse antyder at slike i cellulo- tilnærminger kan være mer effektive enn analysen av rensede krystaller og gi data med høyere oppløsning 20 . Denne protokollen er innePleide å hjelpe forskere ny til protein mikrokrystallografi. Den gir metoder som fokuserer på prøvepreparering og manipulering for røntgendiffraksjon eksperimenter ved en synkrotron stråle linje. To alternativer er foreslått ved å bruke isolerte krystaller for klassisk mikrokrystallografi eller krystallholdige celler sortert ved hjelp av flytcytometri for celluloseanalyse ( figur 1 ).

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			<td height="21" style="height:21px;width:216px;">Sf9 cells</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">SF900-SFM insect medium</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">1L cell culture flask</td>
			<td style="width:127px;">Thermofisher Scientific</td>
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			<td height="42" style="height:42px;width:216px;">Shaking incubator for insect cell culture</td>
			<td style="width:127px;">Eppendorf</td>
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			<td height="21" style="height:21px;width:216px;">50mL conical tubes</td>
			<td style="width:127px;">Falcon</td>
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			<td height="42" style="height:42px;width:216px;">Centrifuge with swing buckets for 50mL tubes</td>
			<td style="width:127px;">Eppendorf</td>
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			<td height="42" style="height:42px;width:216px;">Sonicator equiped with a 19mm probe</td>
			<td>MSE Soniprep 150&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Glass slides</td>
			<td style="width:127px;">Hampton Research</td>
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			<td height="21" style="height:21px;width:216px;">Hemacytometer</td>
			<td style="width:127px;">Sigma-Aldrich</td>
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			<td height="42" style="height:42px;width:216px;">Propidium iodide</td>
			<td style="width:127px;">Thermofisher Scientific</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD Influx cell sorter&#160;</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hampton Heavy atom screens</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Micromesh</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Paper wick</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">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 height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">MX2 microfocus beamline</td>
			<td style="width:127px;">Australian Synchrotron</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">A list of available microfocus beamlines can be found in Boudes et al. (2014) Reflections on the Many Facets of Protein 
			Microcrystallography. Australian Journal of Chemistry 67 (12), 1793&#8211;1806, 
			doi:10.1071/CH14455.</td>
		</tr>
	</tbody>
</table>

microcrystallography of protein crystals and in cellulo diffraction

Tilkomsten av mikrofokus av høy kvalitet på mange synkrotronanlegg har tillatt rutinemessig analyse av krystaller mindre enn 10 μm i sin største dimensjon, som pleide å utgjøre en utfordring. Vi presenterer to alternative arbeidsflyter for strukturbestemmelsen av proteinmikrokrystaller ved røntgenkrystallografi med et spesielt fokus på krystaller vokst in vivo . Mikrokrystallerne ekstraheres enten fra celler ved sonikering og renses ved differensiell sentrifugering, eller analyseres i cellulose etter cellesortering ved hjelp av flytcytometri av krystallholdige celler. Eventuelt blir rensede krystaller eller krystallholdige celler gjennomvåt i tunge atomløsninger for eksperimentell fasering. Disse prøvene fremstilles deretter for diffraksjonseksperimenter på lignende måte ved påføring på en mikromesh-støtte og flash-avkjøling i flytende nitrogen. Vi beskriver kort og sammenligner seriediffraksjon eksperimenter av isolerte mikrokrystaller og krystall-Inneholdende celler ved hjelp av en mikrofokus synkrotron strålelinje for å produsere datasett som er egnet for fasing, modellbygging og forfining. Disse arbeidsflytene er eksemplifisert med krystaller av Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produsert ved infeksjon av insektceller med et rekombinant baculovirus. I denne case-studien er celluloseanalyse mer effektiv enn analyse av rensede krystaller og gir en struktur på ~ 8 dager fra uttrykk til forfining.

En oversikt over begge alternative metoder for strukturbestemmelse ved bruk av in vitro mikrokrystaller presenteres ( figur 1 ). Polyhedra kan lett renses ved sonikering og sentrifugering. På grunn av dens tetthet danner de et lag på bunnen av røret under et lag av rusk som kan fjernes ved pipettering ( figur 3a og 3b ). Prøven blir deretter utsatt for flere runder av sonikering og vasker til et til...

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

En protokoll presenteres for røntgenkrystallografi ved bruk av proteinmikrokrystaller. To eksempler som analyserer in vivo- vokst mikrokrystaller etter rensing eller i cellulose , sammenlignes.

Mikrokrystallografi av proteinkrystaller og<em&gt; I Cellulo</em&gt; Diffraksjon

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

Research

JoVE Journal

Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

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

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

Voksende Protein krystaller med forskjellige dimensjoner bruke automatisert krystallisering kombinert med In Situ dynamisk lysspredning

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

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Måling og tolke oksygen forbruk priser helt Fly hodet segmenter

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Høy følsomhet måling av transkripsjon faktor-DNA Binding slektskap av konkurransedyktige titrering bruker fluorescens mikroskopi

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Analyse av proteinfolding, Transport og nedbrytning i levende celler av radioaktivt puls Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

Helt selvstendig karakterisering og datainnsamling fra krystaller av biologiske makromolekyler

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Anvendelse av Biolayer laboratoriefasiliteter (BLI) for å studere protein-protein interaksjoner i transkripsjon

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Opprette svært spesifikke kjemisk indusert protein Dimerization Systems av trinnvis Phage utvalg av Kombinatorisk single-Domain antistoff bibliotek

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Derivatisering av proteinkrystaller med I3C ved hjelp av tilfeldig mikroseede matrix screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Optimalisering av veksten av endothiapepsinkrystaller for serielle krystallografieksperimenter

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

Microcrystal Electron Diffraction of Small Molecules

Mikrokrystallelektrondiffraksjon av små molekyler

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