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

Ringraziamo il Dr. William Margolin per i commenti. Siamo grati per il sostegno su SerialEM da Drs. David Mastronarde e Chen Xu. DM, BH e JL sono stati sostenuti da Grant R01AI087946 dal National Institute of Allergy e Malattie infettive, Grants R01GM110243 e R01GM107629 presso l&#39;Istituto Nazionale di scienze mediche generali (NIGMS), e Grant AU-1714 dalla Fondazione Welch. Il rivelatore di elettroni diretto è stato finanziato dal National Institutes of Health Award S10OD016279.

1. Minicell Preparazione <ol><li> Per rendere S. minicells flexneri, trasformano 1 ml di plasmide pBS58, che esprime costitutivamente geni divisione cellulare Escherichia coli ftsQ, FTSA, e FtsZ da un basso copy spectinomicina resistente plasmide in 5 microlitri electrocompetent streptomicina resistente sierotipo 5a (M90T-Sm), le cellule di elettroporazione a 2,5 kV per 5 msec a 1 mm cuvette. </li><li> Conservare i campioni minicell a -80 ° C in 15% glicerolo in una provet...

<ol>
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Il metodo di alto-rendimento descritto qui ci ha permesso di elaborare 1.917 crio tilt-serie e produrre oltre 4.500 sub-tomogrammi del intatto S. flexneri injectisome 19. I dati raccolti hanno portato alla caratterizzazione dettagliata di in situ injectisome, tra cui il citoplasmatica di ordinamento complesso. Il metodo è stato utilizzato anche per visualizzare varie cellule mutanti con specifico delezione di componenti proteiche putativi, che ha contribuito a chiarire la composizi...

III sistemi di secrezione di tipo (T3SS) sono fattori di virulenza essenziali per molti patogeni Gram-negativi. Il injectisome, noto anche come il complesso dell&#39;ago, è la macchina centrale T3SS richiesto per traslocazione diretta di proteine ​​effettrici del batterio in cellule ospiti eucariotiche 1, 2. Il injectisome comprende un ago extracellulare, un corpo basale, e un complesso citoplasmatico anche noto come il complesso di smistamento 3. Precedenti studi hanno chiarito le strutture 3-D di injectisomes purificati da Salmonella e Shigella, insieme con le strutture atomiche delle principali proteine ​​del corpo basale 4, 5. Recente in strutture in situ di injectisomes da Salmonella, Shigella, e Yersinia sono stati rivelati da crio-ET 6 , 7. Tuttavia, il complesso citoplasmatico, essenziale per la selezione effettrici e gruppo aghi, non è stata visualizzata in quelle strutture. Cryo-ET è il MOSt tecnica adatta per l&#39;imaging macchinario molecolare presso risoluzione nanometrica all&#39;interno del suo contesto cellulare nativo (in situ). Tuttavia, la risoluzione ottenibile da crio-ET è limitata dalla spessore del provino. Per superare l&#39;inconveniente, abbiamo ripreso injectisomes intatti in una virulenta Shigella flexneri ceppo che è stato geneticamente modificato per produrre minicells abbastanza sottili per crio-ET. Un altro limite di crio-ET è la sensibilità del campione alla radiazione indotta dal fascio di elettroni, che distrugge rapidamente le informazioni ad alta risoluzione nel campione. Come risultato, estremamente basse dosi sono utilizzati per le singole tilt-immagini in modo che una dose adatta può essere distribuito tra l&#39;inclinazione completa serie. Questo riduce notevolmente il rapporto segnale-rumore (SNR) nella ricostruzione finale, il che rende difficile distinguere le caratteristiche strutturali del soggetto dalla grande quantità di rumore nel tomogramma e limita la risoluzione che può essere ottenuta da cryo- ET. Conventielaborazione delle immagini onal quali Fourier e filtri dello spazio reale e giù campionamento può essere utilizzato per aumentare il contrasto, ma a scapito di filtrare gran parte delle informazioni ad alta risoluzione. Recentemente, sub-tomogram media ha consentito di aumentare notevolmente il SNR e successivamente la risoluzione finale in alcuni casi a livelli sub-nanometri 8, 9. Un&#39;analisi più dettagliata dei complessi è reso possibile da computazionalmente estraendo migliaia di sub-tomogrammi contenenti le aree di interesse dalle tomografie originale e quindi allineando e media i sub-tomografie per determinare in strutture complesse in situ con maggiore SNR e una risoluzione più alta. Questi metodi possono essere integrati con approcci genetici per fornire ancora maggiori intuizioni in assemblee macromolecolari e le loro conformazioni dinamici nel contesto cellulare nativo. In generale, decine o centinaia di migliaia di sub-tomogrammi devono essere mediati per determinare altostrutture -Risoluzione in situ. L&#39;acquisizione di un numero sufficiente di tilt serie necessaria per produrre questo grande numero di sotto-tomografie diventa rapidamente un collo di bottiglia. Il conseguente inclinazione della serie sono spesso risente spostamento fascio indotta, fase contraccolpo, e ingrandimento, rotazione e inclinazione difetti, che deve essere risolto per portare l&#39;inclinazione serie in allineamento prima ricostruzione. La serie inclinazione è tipicamente allineato tracciando marker fiduciali oro, che sono tradizionalmente selezionabili manualmente attraverso l&#39;ispezione del tilt-serie, causando ancora un altro collo di bottiglia. Molti pacchetti software sono stati sviluppati per automatiche di acquisizione tilt-serie attraverso microscopi elettronici controllati da computer 10, 11, 12, allineamento tilt-serie e la ricostruzione 13, 14 e sub-tomogramma media 15-18. Poiché questi pacchetti gestiscono operazioni discreti nel flusso di crio-ET, diventa desiderabile costruire un livello più elevato di astrazione nel processo per systemacamente snellire l&#39;intero schema in una singola pipeline. Pertanto, abbiamo sviluppato una libreria wrapper software &quot;tomoauto&quot; progettato per organizzare una serie di questi pacchetti in una singola unità semi-automatico, consentendo funzionamento semplice utente, pur mantenendo la configurazione completa di ciascun componente in modo centralizzato. La biblioteca è open-source, ben documentato, continuamente sviluppato e liberamente disponibili per l&#39;uso, lo sviluppo su misura o una maggiore integrazione per mezzo di un codice sorgente remota repository online (http://github.com/DustinMorado/tomoauto). Questo high-throughput crio-ET gasdotto è stato utilizzato per visualizzare injectisomes intatti a S. minicells flexneri. Un totale di 1.917 tomografie sono stati generati utilizzando questo metodo, rivelando una ad alta risoluzione nella struttura situ della macchina intatta tra cui la piattaforma di smistamento citoplasmatica determinato dal sub-tomogramma media 19. Insieme alla modellistica molecolare di wild-type e mmacchine utant, la nostra pipeline high-throughput fornisce una nuova strada per comprendere la struttura e la funzione del injectisome intatto nel contesto cellulare nativo.

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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.

Campioni di minicells S. flexneri stati raccolti e trattati come indicato in figura 1 utilizzando tomoauto schema seguente pipeline descritto nella figura 2. Tilt-series sono stati raccolti utilizzando SerialEM 10, che permette di high-throughput acquisizione tilt serie nei punti designati da parte dell&#39;utente su mappe montaggio a basso ingrandimento (Figura 3). Micrografie sono...

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 presentiamo un protocollo su come utilizzare high-throughput crio-elettroni tomografia per determinare alta risoluzione in strutture in situ di macchine molecolari. Il protocollo permette di grandi quantità di dati da elaborare, evita i colli di bottiglia comuni e riduce i tempi di inattività delle risorse, permettendo all&#39;utente di concentrarsi su importanti questioni biologiche.

Usando Tomoauto: un protocollo per High-throughput automatizzato Cryo-elettrone Tomografia

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

Microarrays polimerici per Discovery High Throughput di Biomateriali

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

Ricerca

Bioingegneria

High-throughput Protein Expression Generator Using a Microfluidic Platform

High-throughput generatore di espressione delle proteine ​​Utilizzando una piattaforma microfluidica

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

Ad alta velocità, l&#39;estrazione automatizzata di DNA e RNA da campioni clinici utilizzando la tecnologia TruTip su Robot Gestione liquidi comuni

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

Una piattaforma Microfluidic per l&#39;isolamento Single-cell high-throughput e cultura

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

Formazione automatizzato doppio strato lipidico della membrana Utilizzando un Polidimetilsilossano 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 identificazione di batteri Polimeri repellente per dispositivi medici

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

Protocollo generico per l'ottimizzazione della produzione di proteine eterologhe utilizzando tecnologia automatizzata di Microbioreactor

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

Uso di High-Throughput Microbioreactor sistema automatizzato per la produzione del modello IgG1 in cellule CHO

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

Ottimizzazione dei processi mediante microreattori automatizzati ad alta velocità effettiva nella coltivazione delle cellule ovaio dell'ammasso cinese

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

Identificazione ad alto contenuto di velocità effettiva della resistenza a Pseudomonas syringae pv. Pomodoro nel pomodoro utilizzando 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. ...