Name: using tomoauto: a protocol for high-throughput automated cryo-electron tomography
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Wij danken Dr. William Margolin voor commentaar. We zijn dankbaar voor de steun op SerialEM van Drs. David Mastronarde en Chen Xu. DM, BH en JL werden ondersteund door Grant R01AI087946 van het Nationaal Instituut voor Allergie en Besmettelijke Ziekten, subsidies R01GM110243 en R01GM107629 van het National Institute of General Medical Sciences (NIGMS) en Grant AU-1714 van de Welch Foundation. De directe elektron detector werd gefinancierd door de National Institutes of Health Award S10OD016279.

1. Minicel Voorbereiding <ol><li> S. maken flexneri minicellen, transformeren 1 pl plasmide pBS58, die constitutief uitdrukt Escherichia coli celdeling genen FtsQ, ftsA en ftsZ van een low-copy spectinomycine-resistente plasmide in 5 ul elektrocompetente streptomycine resistente serotype 5a (M90T-Sm) cellen door elektroporatie op 2,5 kV voor 5 msec bij 1 mm cuvettes. </li><li> WINKEL minicel monsters bij -80 ° C in 15% glycerol in 1,5 ml microbuisjes cryogeen. Wanneer klaa...

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De high-throughput hier beschreven methode ons in staat om 1917 cryo tilt-serie te verwerken en produceren meer dan 4.500 sub-tomogram van de intacte S. flexneri injectisome 19. De verzamelde gegevens tot de gedetailleerde karakterisatie van in situ injectisome, waaronder het cytoplasmatische sorteren complex. De werkwijze werd eveneens toegepast om verschillende mutantcellen visualiseren specifieke deletie van vermoedelijke eiwitcomponenten, die hielp verhelderen de samens...

Type III secretie systeem (T3SS) zijn essentiële virulentie determinanten voor vele Gram-negatieve pathogenen. De injectisome, ook bekend als de naald complex, is de centrale T3SS machine vereist voor directe translocatie van effector eiwitten van de bacterie in eukaryote gastheercellen 1, 2. De injectisome omvat een extracellulair naald, een basale lichaamstemperatuur, en een cytoplasmatisch complex ook bekende als sorteren complex 3. Eerdere studies hebben opgehelderd 3-D structuur van gezuiverd injectisomes van Salmonella en Shigella, evenals de atomaire structuur van grote basale lichaamseiwitten 4, 5. Recente in situ structuren van injectisomes van Salmonella, Shigella en Yersinia werden onthuld door cryo-ET 6 , 7. De cytoplasmatische complex, essentieel voor effector selectie en naaldsamenstel, is niet zichtbaar in deze structuren. Cryo-ET is het most geschikte techniek voor beeldvorming van moleculaire machines in nanometer resolutie in zijn geboorteland cellulaire context (in situ). Toch is de haalbare resolutie van cryo-ET beperkt door de dikte van het monster. Om het nadeel te overwinnen, we afgebeeld intact injectisomes in een virulente Shigella flexneri stam die genetisch gemodificeerd om minicellen dun genoeg voor cryo-ET produceren. Een andere beperking van cryo-ET is de gevoeligheid van het monster om de straling geïnduceerd door de elektronenbundel, die zeer snel vernietigt de hoge-resolutie-informatie in het monster. Hierdoor zijn zeer lage doses voor individuele tilt-beelden, zodat een geschikte dosis kan worden verdeeld over de volledige tilt-series. Dit vermindert sterk de signaal-ruisverhouding (SNR) in het uiteindelijke reconstructie, wat het moeilijk maakt de structurele kenmerken van het onderwerp van de grote hoeveelheid ruis in het tomogram differentiëren en beperkt de resolutie die kan worden bereikt door cryo- ET. Conventional beeldverwerking zoals Fourier en real-space filters en downsampling kan worden gebruikt om het contrast te verhogen, maar ten koste van het uitfilteren van veel van de hoge-resolutie gegevens. Onlangs heeft sub-tomogram middeling het mogelijk om sterk toenemen van de SNR en vervolgens de uiteindelijke resolutie in sommige gevallen sub-nanometer niveau 8, 9. Een meer gedetailleerde analyse van de complexen mogelijk door computationeel extraheren duizenden sub-tomogram bevattende de aandachtsgebieden van de oorspronkelijke tomogram en vervolgens uitlijnen en het gemiddelde van de sub-tomogram te bepalen in situ complexe structuren met een hogere SNR en een hogere resolutie. Deze werkwijzen kunnen worden geïntegreerd met genetische benaderingen nog meer inzicht verschaffen in macromoleculaire samenstellingen en hun dynamische conformaties in de natieve cellulaire context. In het algemeen tientallen of zelfs honderden duizenden sub-tomogram moeten worden gemiddeld om vast te stellen high-Resolutie structuren in situ. De overname van een voldoende aantal van de tilt-serie die nodig zijn om de productie van dit grote aantal sub-tomogram wordt al snel een bottleneck. De resulterende tilt-serie wordt vaak beïnvloed door-beam geïnduceerde verschuiving, stadium terugslag, alsmede vergroting, rotatie en scheve gebreken, die moeten worden opgelost om de tilt-serie in lijn voorafgaand aan de reconstructie brengen. De tilt-serie wordt meestal uitgelijnd door het bijhouden van goud de vaste markers, die traditioneel met de hand door middel van controle van de tilt-serie zijn geselecteerd, waardoor nog een ander knelpunt. Veel software pakketten zijn ontwikkeld voor geautomatiseerde tilt-serie overname door de computer gestuurde elektronenmicroscopen 10, 11, 12, tilt-serie uitlijning en wederopbouw 13, 14 en sub-tomogram gemiddeld 15-18. Aangezien deze pakketten verwerken discrete activiteiten in de werkstroom van cryo-ET het gewenst wordt om een ​​hoger abstractieniveau bouwen in het proces systematisch stroomlijnen van de gehele regeling in één pijplijn. Daarom hebben we een software wrapper library &quot;tomoauto&quot; bedoeld om een ​​aantal van deze verpakkingen te ordenen in een semi-automatische eenheid, waardoor de gebruiker eenvoudige bediening met behoud van volledige configuratie van elke component op een gecentraliseerde wijze. De bibliotheek is open-source, goed gedocumenteerd, voortdurend ontwikkeld en vrij beschikbaar voor gebruik, op maat ontwikkeling of verdere integratie door middel van een online remote source code repository (http://github.com/DustinMorado/tomoauto). Deze high-throughput cryo-ET pipeline is gebruikt om intact injectisomes in S. visualiseren flexneri minicellen. Een totaal 1.917 tomogram werden gegenereerd met behulp van deze methode, onthullen een hoge resolutie in situ structuur van de intacte machine waaronder het cytoplasmatische sortering platform bepaald door sub-tomogram gemiddeld 19. Samen met moleculaire modellering van wildtype en mutant machines, onze high-throughput pijplijn biedt een nieuwe weg voor de structuur en functie van het intacte injectisome begrijpen de natieve cellulaire context.

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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>
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		<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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			<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.

Monsters van minicellen S. flexneri werden ingezameld en verwerkt zoals getoond in de schematische figuur 1 via tomoauto na de pijpleiding gedetailleerd in figuur 2. Tilt-serie werden verzameld met behulp SerialEM 10, die zorgt voor high-throughput-tilt series verwerving punten door de gebruiker aangewezen lage vergroting montage maps (figuur 3). Microfoto&#39;s werden verzameld met behulp van dosis...

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

We presenteren een protocol over hoe om high-throughput cryo-electron tomography gebruiken om hoge resolutie in situ structuren van moleculaire machines te bepalen. Het protocol maakt grote hoeveelheden gegevens te verwerken, vermijdt gemeenschappelijke resource bottlenecks en vermindert stilstand, zodat de gebruiker zich concentreren op belangrijke biologische vragen.

Met behulp van Tomoauto: Een protocol voor 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 ...

Research

JoVE Journal

Bioengineering

Polymer Microarrays for High Throughput Discovery of Biomaterials

Polymeer Microarrays voor High Throughput Ontdekking van biomaterialen

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 eiwit expressie Generator Met behulp van een Microfluïdische 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

High-throughput, geautomatiseerde extractie van DNA en RNA uit klinische monsters met behulp TruTip Technology op Common Liquid Handling 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

Een microfluïdische platform voor High-throughput Single-cell Isolatie en Cultuur

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

Geautomatiseerde lipidebilaagmembraan Vorming Met behulp van een 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

High-throughput Identificatie van bacteriën Repellent Polymers for Medical Devices

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

Generic-Protocol voor optimalisatie van heterologe eiwitproductie met behulp van geautomatiseerde Microbioreactor technologie

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

Gebruik van High-Throughput geautomatiseerd Microbioreactor-systeem voor de productie van Model IgG1 in CHO cellen

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

Proces optimalisatie met behulp van high throughput geautomatiseerde micro-bioreactoren in de Chinese Hamster Eierstok celteelt

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 Identificatie van resistentie tegen Pseudomonas syringae pv. Tomaat in Tomaat met seedling Flood Assay

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