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 taknemmelige for den støtte på SerialEM fra Drs. David Mastronarde og Chen Xu. DM, BH og JL blev støttet af Grant R01AI087946 fra National Institute of Allergy og smitsomme sygdomme, Grants R01GM110243 og R01GM107629 fra National Institute of General Medical Sciences (NIGMS), og Grant AU-1714 fra Welch Foundation. Den direkte elektron detektor blev finansieret af National Institutes of Health Award S10OD016279.

1. Minicell Forberedelse <ol><li> For at gøre S. flexneri miniceller, transformere 1 pi plasmid pBS58, som konstitutivt udtrykker Escherichia coli celledeling gener ftsQ, ftsA og ftsZ fra en lav-kopi spectinomycin-resistent plasmid i 5 pi elektrokompetente streptomycin-resistente serotype 5a (M90T-Sm) celler ved elektroporering ved 2,5 kV i 5 ms i 1 mm kuvetter. </li><li> Opbevar minicelle prøver ved -80 ° C i 15% glycerol i en 1,5 ml mikrorør. Kryogen Når klar til brug, skrabe...

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Den her beskrevne high-throughput metode muligt for os at behandle 1917 kryo tilt-serier og producerer over 4.500 sub-tomogrammer af den intakte S. flexneri injectisome 19. De indsamlede data førte til detaljeret karakterisering af in situ injectisome, herunder den cytoplasmatiske sortering kompleks. Metoden blev også anvendt til at visualisere flere mutantceller specifikke deletion af formodede proteinkomponenter, som hjalp belyse sammensætningen af ​​sorteringsanlægget pla...

Type III sekretionssystemer (T3SS) er vigtige virulensdeterminanter for mange Gram-negative patogener. Den injectisome, også kendt som nålen komplekset, er den centrale T3SS maskinen kræves til direkte translokation af effektorceller proteiner fra bakterien i eukaryote værtsceller 1, 2. Den injectisome omfatter et ekstracellulært nål, en basal krop, og et cytoplasmatisk kompleks også kendt som sortering komplekse 3. Tidligere undersøgelser har belyst 3-D strukturer af oprensede injectisomes fra Salmonella og Shigella, sammen med de atomare strukturer af store basale kropsproteiner 4, 5. Nylige in situ strukturer injectisomes fra Salmonella, Shigella og Yersinia blev afsløret ved cryo-ET 6 7. Men den cytoplasmatiske komplekse, afgørende for effektor udvælgelse og nåleaggregatet, er ikke blevet visualiseret i disse strukturer. Cryo-ET er MOSt egnet teknik til billeddannelse molekylær maskiner på nanometer opløsning inden for sit oprindelige cellulære kontekst (in situ). Ikke desto mindre er den opnåelige beslutning cryo-ET begrænset af prøven tykkelse. For at overvinde den ulempe, vi afbildes intakte injectisomes i en virulent Shigella flexneri stamme, der var genetisk modificeret til at producere miniceller tynde nok til kryo-ET. En anden begrænsning ved kryo-ET er følsomheden af ​​prøven for strålingen induceret af elektronstrålen, som meget hurtigt ødelægger information med høj opløsning i prøven. Som et resultat, er yderst lave doser anvendes til individuelle tilt-billeder, så en passende dosis kan fordeles blandt de full tilt-serien. Dette sænker signal-til-støj-forholdet (SNR) i den endelige genopbygning, hvilket gør det vanskeligt at skelne de strukturelle træk af emnet fra den store mængde af støj i tomogram og begrænser opløsning, der kan opnås ved fryselagring stærkt ET. ConventiOnal billedbehandling såsom Fourier og real-space-filtre samt ned prøvetagning kan bruges til at øge kontrasten, men på bekostning af bortfiltrere meget af oplysninger den høje opløsning. For nylig har sub-tomogram gennemsnitsberegning gjort det muligt i høj grad at øge SNR og efterfølgende den endelige opløsning i nogle tilfælde til sub-nanometer niveauerne 8, 9. En mere detaljeret analyse af komplekser er gjort mulig ved beregningsmæssigt udvinder tusindvis af sub-tomogrammer indeholder de områder af interesse fra de oprindelige tomogrammer og derefter tilpasse og gennemsnit sub-tomogrammer at bestemme in situ komplekse strukturer med højere SNR og højere opløsning. Disse metoder kan integreres med genetiske metoder til at opnå en endnu bedre indsigt i makromolekylære forsamlinger og deres dynamiske konformationer i den native cellulære kontekst. Generelt skal gennemsnit for at bestemme høj snesevis eller endda hundredvis af tusinde sub-tomogrammer-Opløsning strukturer i stedet. Købet af et tilstrækkeligt antal tilt-serien er nødvendig for at producere denne store antal sub-tomogrammer hurtigt bliver en flaskehals. Den resulterende tilt-serien er ofte påvirket af stråle-induceret skift, scene slør, samt forstørrelse, rotation og skæve defekter, som skal løses for at bringe tilt-serien på linje forud for genopbygning. Tilt serien er typisk rettet ind ved at spore guld referencemærker markører, der traditionelt er valgt manuelt via inspektion af tilt-serien, der forårsager endnu en flaskehals. Mange softwarepakker er blevet udviklet til automatiseret tilt-serien erhvervelse gennem computerstyrede elektronmikroskoper 10, 11, 12, tilt-serien tilpasning og genopbygning 13, 14 og sub-tomogram gennemsnit 15-18. Da disse pakker håndtere diskrete operationer i arbejdsgangen af ​​kryo-ET, bliver det ønskeligt at opbygge et højere abstraktionsniveau i processen til Systemamatisk strømline hele ordningen i en enkelt rørledning. Derfor har vi udviklet en software wrapper bibliotek &quot;tomoauto&quot; designet til at organisere en række af disse pakker i en enkelt halvautomatisk enhed, der giver mulighed for enkel brugerbetjening og samtidig opretholde fuld konfiguration af hver komponent i en centraliseret måde. Biblioteket er open source, veldokumenteret, løbende udviklet og frit tilgængelige til brug, skræddersyet udvikling eller yderligere integration ved hjælp af en online remote kildekode repository (http://github.com/DustinMorado/tomoauto). Denne high-throughput kryo-ET rørledningen er blevet anvendt til at visualisere intakte injectisomes i S. flexneri miniceller. I alt 1.917 tomogrammer blev genereret ved hjælp af denne metode, afslører en høj opløsning in situ struktur intakt maskine inklusive cytoplasmatiske sortering platform bestemt af sub-tomogram gennemsnit 19. Sammen med molekylær modellering af vildtype og mutant maskiner, vores high-throughput pipeline tilvejebringer en ny vej til at forstå strukturen og funktionen af ​​det intakte injectisome i det native cellulære kontekst.

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	<tbody>
		<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>
		</tr>
		<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>
		</tr>
		<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>
			<td style="width:164px;"></td>
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		<tr height="63">
			<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 af miniceller S. flexneri blev indsamlet og behandlet som vist i den skematiske figur 1 under anvendelse af tomoauto følge rørledningen vist detaljeret i figur 2. Tilt-serien blev indsamlet ved hjælp af SerialEM 10, som giver mulighed for high-throughput tilt-serien erhvervelse ved punkter, der er udpeget af brugeren på lav-forstørrelse montage kort (figur 3). Mikrograf...

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 præsenterer en protokol om, hvordan man udnytter high-throughput cryo-elektron tomografi til at bestemme høj opløsning in situ strukturer af molekylære maskiner. Protokollen tillader store mængder data, der skal behandles, undgår fælles flaskehalse og reducerer ressource nedetid, så brugeren kan fokusere på vigtige biologiske spørgsmål.

Brug Tomoauto: En protokol til High-throughput Automatiseret 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

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Bioengineering

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

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

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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 ...

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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

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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 protokol for optimering af heterolog Protein produktion ved hjælp af automatiserede Microbioreactor teknologi

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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

High-Throughput Identifikation af resistens over for Pseudomonas syringae pv. Tomat i tomat ved hjælp af sætteplante Oversvømmelse Assay

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