We thank Dr. William Margolin for comments. We are grateful for the support on SerialEM from Drs. David Mastronarde and Chen Xu. D.M., B.H. and J.L. were supported by Grant R01AI087946 from the National Institute of Allergy and Infectious Diseases, Grants R01GM110243 and R01GM107629 from the National Institute of General Medical Sciences (NIGMS), and Grant AU-1714 from the Welch Foundation. The direct electron detector was funded by National Institutes of Health Award S10OD016279.

1. Minicell Preparation
<ol>
	<li>To make S. flexneri minicells, transform 1 &#181;l of plasmid pBS58, which constitutively expresses Escherichia coli cell division genes ftsQ, ftsA, and ftsZ from a low-copy spectinomycin-resistant plasmid into 5 &#181;l electrocompetent Streptomycin-resistant serotype 5a (M90T-Sm) cells by electroporation at 2.5 kV for 5 msec in 1 mm cuvettes.</li>
	<li>Store minicell samples at -80 &#176;C in 15% glycerol in a 1.5 ml cryogenic microtube. When ready...

<ol>
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The high-throughput method described here enabled us to process 1,917 cryo tilt-series and produce over 4,500 sub-tomograms of the intact S. flexneri injectisome19. The collected data led to the detailed characterization of in situ injectisome, including the cytoplasmic sorting complex. The method was also utilized to visualize several mutant cells with specific deletion of putative protein components, which helped elucidate the composition of the sorting platform of the injectisome....

Type III secretion systems (T3SS) are essential virulence determinants for many Gram-negative pathogens. The injectisome, also known as the needle complex, is the central T3SS machine required for direct translocation of effector proteins from the bacterium into eukaryotic host cells1, 2. The injectisome comprises an extracellular needle, a basal body, and a cytoplasmic complex also known as the sorting complex3. Previous studies have elucidated 3-D structures of purified injectisomes from Salmonella and Shigella, along with the atomic structures of major basal body proteins4, 5. Recent in situ structures of injectisomes from Salmonella, Shigella, and Yersinia were revealed by cryo-ET6, 7. However, the cytoplasmic complex, essential for effector selection and needle assembly, has not been visualized in those structures.
Cryo-ET is the most suitable technique for imaging molecular machinery at nanometer resolution within its native cellular context (in situ). Nevertheless, the achievable resolution by cryo-ET is limited by specimen thickness. To overcome the drawback, we imaged intact injectisomes in a virulent Shigella flexneri strain that was genetically modified to produce minicells thin enough for cryo-ET. Another limitation of cryo-ET is the sensitivity of the sample to the radiation induced by the electron beam, which very quickly destroys the high-resolution information in the sample. As a result, extremely low doses are used for individual tilt-images so that a suitable dose can be distributed amongst the full tilt-series. This greatly lowers the signal-to-noise ratio (SNR) in the final reconstruction, which makes it difficult to differentiate the structural features of the subject from the large amount of noise in the tomogram and limits the resolution that can be achieved by cryo-ET. Conventional image processing such as Fourier and real-space filters as well as down sampling can be used to increase contrast, but at the expense of filtering out much of the high-resolution information. Recently, sub-tomogram averaging has made it possible to greatly increase the SNR and subsequently the final resolution in some cases to sub-nanometer levels8, 9. A more detailed analysis of complexes is made possible by computationally extracting thousands of sub-tomograms containing the areas of interest from the original tomograms and then aligning and averaging the sub-tomograms to determine in situ complex structures with higher SNR and higher resolution. These methods can be integrated with genetic approaches to provide even greater insights into macromolecular assemblies and their dynamic conformations in the native cellular context.
In general, tens or even hundreds of thousand sub-tomograms need to be averaged in order to determine high-resolution structures in situ. The acquisition of a sufficient number of tilt-series needed to produce this large number of sub-tomograms quickly becomes a bottleneck. The resulting tilt-series are often affected by beam-induced shift, stage backlash, as well as magnification, rotation and skew defects, which must be solved to bring the tilt-series into alignment prior to reconstruction. The tilt series is typically aligned by tracking gold fiducial markers, which are traditionally selected manually through inspection of the tilt-series, causing yet another bottleneck. Many software packages have been developed for automated tilt-series acquisition through computer-controlled electron microscopes10, 11, 12, tilt-series alignment and reconstruction13, 14 and sub-tomogram averaging15-18. As these packages handle discrete operations in the workflow of cryo-ET, it becomes desirable to build a higher level of abstraction into the process to systematically streamline the entire scheme into a single pipeline. Therefore, we developed a software wrapper library &#34;tomoauto&#34; designed to organize a number of these packages into a single semi-automated unit, allowing for simple user operation while maintaining full configuration of each component in a centralized manner. The library is open-source, well documented, continually developed and freely available for use, tailored development or further integration by means of an online remote source code repository (http://github.com/DustinMorado/tomoauto).
This high-throughput cryo-ET pipeline has been utilized to visualize intact injectisomes in S. flexneri minicells. A total of 1,917 tomograms were generated using this method, revealing a high-resolution in situ structure of the intact machine including the cytoplasmic sorting platform determined by sub-tomogram averaging19. Together with molecular modeling of wild-type and mutant machines, our high-throughput pipeline provides a new avenue to understand the structure and function of the intact injectisome in the native cellular context.

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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>
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			<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>
		</tr>
		<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.

Samples of minicells S. flexneri were collected and processed as showed in the schematic Figure 1 using tomoauto following the pipeline detailed in Figure 2. Tilt-series were collected using SerialEM10, which allows for high-throughput tilt-series acquisition at points designated by the user on low-magnification montage maps (Figure 3). Micrographs were collected using dose-fractionation mode on a dire...

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 present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

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

The overall goal of this procedure is to help users establish a high-throughput collection and processing pipeline of cryo-electron tomography. This method can help answer key questions in the cryo-electron tomorgraphy field, such as:how do I handle data collected with new generation detectors, and process a huge amount of data produced by automated tilt-series collection software? The main advantage with this technique is that the user can configure multiple software packages in a unifying manner, to finding an efficient pipeline for automated tilt-series processing, allowing for more time to be spent on tomogram analysis.Generally, individuals new to cryo-ET will struggle because many tedious steps are required to handle massive data generated from the electron microscope. We first had the idea for this development five years ago when we had to manually go through hundreds of steps to get a 3D reconstruction of one bacteria. In advance, consult the text protocol on preparing the Shigella flexneri minicells.Then, prepare the electron microscopy grids as explained by an existing JoVE publication and also documented in the text protocol. After having loaded the EM stage with the collection of minicells embedded in vitrified ice, start collecting low-magnification maps for the tilt-series. While viewing the fluorescent screen at low magnification, of about 2300x, find grid squares that are acceptable to image.This location should contain thin ice and the subject of interest with no contaminants. Next, adjust the view to the center of the grid square, and click on the Add Stage Position button to save the location. Then, repeat the process until all the acceptable grid squares have had their location saved.The next step begins with opening a new montage file. In the setup dialog, set the X and Y so that the entire grid square is captured. For a standard, 200 mesh grid, make a 10 by 10 space with a high Binning value, like eight.Also toggle the move stage instead of shifting image option and the skip correlations used to align pieces option. Now, in the Navigator window, select the first stage position to be acquired using the Acquire toggle. Continue doing this for each stage position.Next, under the Navigator menu, click on Acquire at Points. In the dialog box that opens up, toggle acquire map image and rough eucentricity. Untoggle all the other options.Then, click proceed, and a montage will be made for each stage position. Continue working with the SerialEM software. In the Navigator, select the acquired maps, and click on Load Map.Then, click on Add Points, and select the points on the map where the tilt-series should be acquired. After making the selections, click Stop Adding Points and repeat the process with each collected map. Next, open the parameters under the Camera menu, and set the Focus, Trial, and Record modes.The option to record dose-fractionated data may be of special interest, and that can be specified under the Record options. To proceed, select one of the points in the currently viewed map, and toggle the tilt-series option in the Navigator window. In the dialog box that opens, choose the parameters for the tilt-series.Then, repeat the process of toggling tilt-series option for each of the selected points in the map. Next, from the Navigator start the Acquire at Points option. Under the dialog box, toggle the Preliminary Tasks, Realign to item, Autofocus, and rough eucentricity.Then, set the Primary Task to acquire tilt-series, and choose close column vals at end to close the column when all the points have been collected. When the process is executed, a tilt-series will be collected at each point toggled for tilt-series collection in each map. With the original tilt-series, the output log from SerialEM, and the individual dose-fractionated images all in the current working directory, in a terminal, execute the following command to remove the beam-induced motion artifact.Proceed with aligning and reconstructing the tilt-series. Type in tomoauto command in the terminal as follows. To the command, append the filename of the tilt-series to process, followed by the fiducial diameter in nanometers.If CTF processing is not desired, simply omit the option from the command. This command is the main use of tomoauto, allowing users to execute all the processes involved in aligning a tilt-series, which originally required manual user intervention, in a single command, allowing users to quickly script batch processing. Now, using the 3dmod command, look over the aligned tilt-series for any glaring errors.Here filename is the name of the tilt-series without suffix. Then, inspect the estimated CTF with the command shown here. Again, filename is the name of the tilt-series without suffix.Also, in the output log produced by the tomoauto command, check the residual error of the alignment. This is a quantitative measure of the alignment's overall quality. If the alignment is acceptable, then proceed with computing the reconstruction.This command is set to correct the CTF, and erase the fiducial markers used in alignment from the tilt-series. To skip the visual inspection, and go directly to the reconstruction, use this command. Tomoauto has many accessible options.One in particular is the ability to include local configurations described by an auxiliary file, according to the tomoauto documentation. Include this file's name in the commandline dialog using the local configuration option before appending the tilt-series filename. The ability to create local configuration files is the most powerful feature of tomoauto, allowing users to fully configure every command used, tuning execution to specific or difficult data sets while maintaining the automated processing.Within the test protocol are additional instructions on how to execute sub-tomogram averaging. Following the described protocols, two aligned tilt-series were made with tomoauto. The fine alignment is calculated from a model that ideally defines fiducial marker coordinates.At 50 degrees, one tilt-series tracked the gold particles correctly, indicating an acceptable alignment. While, in the case of the second tilt-series, several model points, shown in red, strayed from their corresponding gold marker, illustrating the model did not produce suitable alignment. When tilt-series are collected, tomoauto successfully aligns 80 to 90%of the collected tilt-series.After reconstruction, the final tomogram is a 3D volume of the image sample. This can then be used for cellular annotation by segmentation, or sub-tomogram averaging to obtain higher resolution information of the molecular machinery within the sample. The 2.7 nanometers sub-tomogram average of the attached Shigella flexneri deposited in the EMDB shows the large improvement of this technique over viewing the injectisome in a single tomogram.After watching this video, you should have a good understanding of how to collect and process cryo-electron tomography data using SerialEM and tomoauto in an automated, high-throughput manner. Once mastered, this technique can be used to collect almost 100 tilt-series in a day, if it is performed properly, and align a tilt-series in 20 minutes. While attempting this procedure, it is important to remember to always check the quality of tilt-series alignment.If a set of tilt-series does not align well, try aligning manually, and transfer the parameters to a local configuration. Following this procedure, other methods like sub-tomogram averaging can be performed in order to answer additional questions, like determining the higher-resolution structure of macro-molecular complexes. After its development, this technique paved the way for researchers in the field of cryo-EM to explore high-resolution in situ structures in bacteria, viruses, and other organisms.

using-tomoauto-protocol-for-high-throughput-automated-cryo-electron

Research

JoVE Journal

Bioengineering

10.8K visualizações.  University of Texas Health Science Center at Houston. The overall goal of this procedure is to help users establish a high-throughput collection and processing pipeline of cryo-electron tomography. This method can help answer key questions in the cryo-electron tomorgraphy field, such as:how do I handle data collected with new generation detectors, and process a huge amount of data produced by automated tilt-series collection software? The main advantage with this technique is that the user can configure multiple software packages in a unifying m...