Name: using tomoauto: a protocol for high-throughput automated cryo-electron tomography
Uploaded: 2016-01-30T15:00:00
Description: Watch this Scientific Journal Video about Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography at JoVE.com

Vi takker Dr. William Margolin for kommentarer. Vi er takknemlige for støtten på SerialEM fra legene. David Mastronarde og Chen Xu. DM, BH og JL ble støttet av Grant R01AI087946 fra National Institute of Allergy og smittsomme sykdommer, Grants R01GM110243 og R01GM107629 fra National Institute of General Medical Sciences (NIGMS), og Grant AU-1714 fra Welch Foundation. Den direkte elektrondetektor ble finansiert av National Institutes of Health Award S10OD016279.

1. Power reduction Forberedelse <ol><li> For å gjøre S. flexneri minicells, forvandle 1 mL av plasmid pBS58, som konstitutivt uttrykker Escherichia coli celledeling gener ftsQ, ftsA, og ftsZ fra en lav-kopi spectinomycin motstandsdyktig plasmid inn 5 ul elektrokompetente streptomycin bestandig serotype 5a (M90T-Sm) celler ved elektroporering på 2,5 kV for 5 ms i 1 mm kyvetter. </li><li> Oppbevar minicellen prøver ved -80 ° C i 15% glycerol i en 1,5 ml kryogenisk mikrorør. Når...

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The high-throughput metoden beskrevet her gjorde oss i stand til å behandle 1917 cryo tilt-serien og produserer over 4500 under tomograms av intakt S. flexneri injectisome 19. De innsamlede data førte til detaljert karakterisering av in situ injectisome, inkludert cytoplasmatiske sortering kompleks. Fremgangsmåten ble også anvendt for å visualisere flere mutante celler med spesifikk delesjon av antatte proteinkomponenter, som bidro til å klargjøre sammensetningen av sortering...

Type III sekresjon systemer (T3SS) er viktige virulens determinants for mange Gram-negative patogener. Den injectisome, også kjent som p-komplekset, er den sentrale T3SS maskinen som er nødvendig for direkte translokasjon av effektor-proteiner fra bakterien i eukaryote vertsceller 1, 2. Den injectisome omfatter en ekstracellulær nål, en basal legeme, og en cytoplasmisk kompleks også kjent som sortering kompleks tre. Tidligere studier har belyst 3-D strukturer av rensede injectisomes fra Salmonella og Shigella, sammen med atomstruktur i større basal kroppsproteiner 4, 5. Nylige in situ strukturer av injectisomes fra Salmonella, Shigella, og Yersinia ble åpenbart av kryo-ET 6 , 7. Imidlertid cytoplasmatiske kompleks, avgjørende for valg effektor og nålanordning har ikke blitt visualisert i disse strukturer. Cryo-ET er most egnet teknikk for å avbilde molekylære maskiner på nanometer oppløsning innenfor sitt opprinnelige cellulære kontekst (in situ). Likevel er den oppnåelige oppløsningen av kryo-ET begrenset av prøvetykkelse. For å overvinne den ulempen, vi avbildes intakte injectisomes i en ondartet Shigella flexneri stamme som ble genmodifisert til å produsere minicells tynne nok for kryo-ET. En annen begrensning av cryo-ET er følsomheten av prøven for stråling indusert av elektronstrålen, som meget raskt ødelegger informasjon med høy oppløsning i prøven. Som et resultat, er ekstremt lave doser som brukes for individuelle tilt-bilder, slik at en passende dosering kan bli fordelt på hele tilt-serien. Dette reduserer i stor grad signal-til-støy-forhold (SNR) i den endelige rekonstruksjon, noe som gjør det vanskelig å skille de strukturelle trekk av emnet fra den store mengden av støy i tomogram og begrenser oppløsningen som kan oppnås ved cryo- ET. Conventional bildebehandling for eksempel Fourier og real-space filtre, så vel som nedover sampling kan brukes til å øke kontrasten, men på bekostning av å filtrere ut mye av den informasjon med høy oppløsning. Nylig har sub-tomogram midling gjort det mulig å øke den SNR og deretter den endelige oppløsning i noen tilfeller til under nanometer nivåer 8, 9. En mer detaljert analyse av kompleksene er gjort mulig ved beregningsmessig å ekstrahere flere tusen under tomograms innehold de områder av interesse fra den opprinnelige tomograms og deretter samkjøre og gjennomsnitt under tomograms å bestemme in situ komplekse strukturer med høyere SNR og høyere oppløsning. Disse metodene kan integreres med genetiske metoder for å gi enda større innsikt i makromolekyl forsamlinger og deres dynamiske conformations i morsmobil sammenheng. Generelt titalls eller hundrevis av tusen under tomograms må i gjennomsnitt for å bestemme høy-Oppløsning strukturer in situ. Oppkjøpet av et tilstrekkelig antall tilt-serien nødvendig for å produsere dette stort antall under tomograms blir fort en flaskehals. Den resulterende vippeserien er ofte påvirket av stråleinduserte skift, trinnet tilbakeslag, samt forstørrelse, rotasjon og skew defekter, som må løses for å bringe vippe-serien til innretting før rekonstruksjon. Tilt serien er vanligvis justert av sporingsgull fiducial markører, som er tradisjonelt valgt manuelt gjennom inspeksjon av tilt-serien, forårsaker enda en flaskehals. Mange programvarepakker som er utviklet for automatisk tilt-serie oppkjøp gjennom datastyrte elektronmikroskop 10, 11, 12, tilt-serien justering og gjenoppbygging 13, 14 og sub-tomogram gjennomsnitt 15-18. Ettersom disse pakkene håndtere adskilte operasjoner i arbeidsflyten av cryo-ET, blir det ønskelig å bygge opp et høyere abstraksjonsnivå inn i prosessen for å systematisk effektivisere hele ordningen til en enkelt rørledning. Derfor har vi utviklet en programvare wrapper bibliotek &quot;tomoauto&quot; utviklet for å organisere en rekke av disse pakkene i en enkelt semi-automatisert enhet, noe som åpner for enkel bruker drift samtidig opprettholde fulle konfigurasjon av hver komponent i en sentralisert måte. Biblioteket er open-source, godt dokumentert, kontinuerlig utviklet og fritt tilgjengelig for bruk, skreddersydd utvikling eller ytterligere integrering ved hjelp av en elektronisk fjern kildekode repository (http://github.com/DustinMorado/tomoauto). Dette high-throughput cryo-ET rørledningen har blitt benyttet for å visualisere intakte injectisomes i S. flexneri minicells. Totalt 1,917 tomograms ble generert ved hjelp av denne metoden, avslører en høy oppløsning in situ strukturen intakt maskin inkludert cytoplasma sortering plattformen bestemmes av sub-tomogram gjennomsnitt 19. Sammen med molekylær modellering av villtype og mutant maskiner, gir vår høy gjennomstrømming rørledning en ny vei for å forstå strukturen og funksjonen til intakt injectisome i den innfødte cellular sammenheng.

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		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Glycerol</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:116px;">G9012</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Tyrptic Soy Broth</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:116px;">22092</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Spectinomycin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:116px;">S0692</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:160px;">Electroporation Apparatus</td>
			<td style="width:211px;">Bio-rad</td>
			<td>165-2100</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">1 mm Cuvette</td>
			<td style="width:211px;">BTX</td>
			<td>45-0124</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">1.5 mL Cryogenic Tube</td>
			<td style="width:211px;">Thermoscientific</td>
			<td>5000-1020</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:160px;">1.5 mL Microcentrifuge Tube</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td>Z336769</td>
			<td style="width:164px;"></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:160px;">Holey Carbon Grids</td>
			<td style="width:211px;">Quantifoil 
			(Electron Microscopy Sciences)</td>
			<td style="width:116px;">Q2100CR2</td>
			<td style="width:164px;">R2/2 200 Cu</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:160px;">Glow Discharge Device</td>
			<td style="width:211px;">In-House</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">Commercial Alternative Available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:160px;">Vacuum Desiccator</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:116px;">Z119016&#160;</td>
			<td style="width:164px;">Used in In-House Glow Discharge Device</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:160px;">High-Frequency Generator</td>
			<td style="width:211px;">Electro-Technic Products</td>
			<td style="width:116px;">BD-10A</td>
			<td style="width:164px;">Used in In-House Glow Discharge Device.&#160; CAUTION: This device generates high voltages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Centrifuge</td>
			<td style="width:211px;"></td>
			<td style="width:116px;"></td>
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			<td height="63" style="height:63px;width:160px;">Forceps</td>
			<td style="width:211px;">Dumont 
			(Electron Microscopy Sciences)</td>
			<td style="width:116px;">72705-D</td>
			<td style="width:164px;">Style 5 Anti-magnetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Colliodal Gold</td>
			<td style="width:211px;">Aurion</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">BSA 10nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Filter Paper</td>
			<td style="width:211px;">Whatman</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">#2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Ethane&#160;</td>
			<td style="width:211px;">Matheson Tri-Gas</td>
			<td style="width:116px;">UN1035</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Nitrogen</td>
			<td style="width:211px;">Matheson Tri-Gas</td>
			<td style="width:116px;">UN1977</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:160px;">Plunger Device</td>
			<td style="width:211px;">In-House</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">Commercial Alternative Available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:160px;">Cryogenic Grid Storage Box</td>
			<td style="width:211px;">Electron Microscopy Sciences</td>
			<td style="width:116px;">71166-30</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:160px;">Transmission Electron Microscope</td>
			<td style="width:211px;">FEI</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">Tecnai Polara F30 
			(300 KeV)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:160px;">Direct Detection Device Camera</td>
			<td style="width:211px;">Gatan</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">K2 Summit</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:160px;">Tomogram Acquisiton Software</td>
			<td style="width:211px;">SerialEM</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">http://bio3d.colorado.eud/SerialEM Alternatives: UCSF Tomography, Leginon, FEI Batch Tomography</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:160px;">Beam-induced Motion Correction Software</td>
			<td style="width:211px;">MOTIONCORR</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">http://cryoem.ucsf.edu/software/driftcorr.html Requires &#62;2GB Nvidia GPU</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:160px;">Tilt-Series Alignment Software</td>
			<td style="width:211px;">IMOD</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">http://bio3d.colorado.edu/IMOD Alternatives: XMIPP, Protomo</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:160px;">Automatic Fiducial Marker Modelling Software</td>
			<td style="width:211px;">IMOD</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">Alternatives: RAPTOR (Included in IMOD0 
			(Usable in tomoauto)</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:160px;">CTF Determination Software</td>
			<td style="width:211px;">IMOD</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">Alternatives: CTFFIND http://grigoriefflab.janelia.org/ctf 
			(Usable in tomoauto)</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:160px;">Tilt-Series Reconstruction Software</td>
			<td style="width:211px;">tomo3d</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">https://sites.google.com/site/3demimageprocessing/tomo3d Alternatives: IMOD, XMIPP http://xmipp.cnb.csic.es , Protomo</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:160px;">Tilt-Series Automated Processing Software</td>
			<td style="width:211px;">tomoauto</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">https://github.com/DustinMorado/tomoauto</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:160px;">Particle Picking Software</td>
			<td style="width:211px;">i3</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">http://www.electrontomography.org Alternatives: IMOD</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:160px;">Subvolume Averaging Software</td>
			<td style="width:211px;">i3</td>
			<td style="width:116px;"></td>
			<td style="width:164px;">Alternatives: PEET http://bio3d.colorado.edu/PEET, Dynamo https://dynamo.bioz.unibas.ch , PyTom http://pytom.org</td>
		</tr>
	</tbody>
</table>

using tomoauto: a protocol for high-throughput automated cryo-electron tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

Prøver av minicells S. flexneri ble samlet og behandlet som vist i den skjematiske figur 1 under anvendelse av følgende tomoauto rørledningen detaljert i figur 2. Tilt-serien ble samlet inn ved hjelp SerialEM 10, som gir mulighet for høy gjennomstrømming tilt-serie oppkjøp på punkter utpekt av brukeren på lav forstørrelse montage kart (figur 3). Mikrografer ble samlet inn ved hjelp dose-fra...

Watch this Scientific Journal Video about Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography at JoVE.com

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Vi presenterer en protokoll på hvordan man kan utnytte høy gjennomstrømming cryo-electron tomography å bestemme høy oppløsning in situ strukturer av molekylære maskiner. Protokollen tillater store mengder data som skal behandles, unngår vanlige flaskehalser og reduserer ressurs nedetid, slik at brukeren kan fokusere på viktige biologiske spørsmål.

Bruke Tomoauto: En protokoll for høy gjennomstrømming Automated Cryo-elektron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

Research

JoVE Journal

Bioengineering

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

High-throughput Protein Expression Generator Ved hjelp av en Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

Høy gjennomstrømning, Automated Utvinning av DNA og RNA fra kliniske prøver ved hjelp TruTip Technology på Common Væskehåndtering Robots

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

En mikrofluid plattform for High-throughput encellede Isolering og kultur

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

Automatisert lipid bilaget Membran Dannelse Ved hjelp av en Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Høy throughput identifisering av bakterier Repellent Polymers for medisinsk utstyr

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

Generisk protokoll for optimalisering av Heterologous Protein produksjonen ved hjelp av automatisert Microbioreactor teknologi

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Bruk av høy gjennomstrømming automatisert Microbioreactor System for produksjon av modellen IgG1 i CHO celler

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Prosessoptimalisering ved hjelp av høy gjennomstrømning automatiserte mikrobioreaktorer i kinesisk hamster eggstokk celle dyrking

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Høy-gjennomstrømning identifikasjon av motstand mot Pseudomonas syringae pv. Tomat i tomat ved hjelp av frøplanteflomanalyse

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...