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 for use, scrape approximately 5 &#956;l of cells from the unthawed microtube using a pipette tip and suspend the cells in 4 ml tryptic soy broth with spectinomycin added to 100 &#956;g/ml concentration. Grow O/N at 37 &#176;C.</li>
	<li>Pipette 2 ml of the culture from 1.2 into 200 ml tryptic soy broth with spectinomycin again added to 100 &#956;g/ml concentrations. Grow at 37 &#176;C to late log phase.</li>
	<li>To enrich minicells, centrifuge 200 ml of the culture from 1.3 at 1,000 x g for 5 min. Carefully pour the supernatant fraction into a new centrifuge tube and centrifuge at 20,000 x g for 10 min. Carefully pour off and discard the supernatant fraction, and gently mix the pellet with the remaining liquid using a pipette tip and transfer approximately 100 &#956;l of the pellet mixture to a 1.5 ml microcentrifuge tube.</li>
</ol>
2. EM Grid Preparation
<ol>
	<li>Place a R2/2 holey carbon film 200-mesh copper grid carbon side up on a glass slide. 
	NOTE: A R2/2 200-mesh grid is selected to maximize the number of tilt-series that can be set to acquire in a single grid square while still supporting the specimen and placing the edge of the carbon film in the field of the camera at the desired magnification. Finer mesh grids and smaller holey carbon films such as R1.2/1.3 400-mesh can be used for samples imaged at higher magnification; larger holey carbon films such as R3.5/1 200-mesh can be used for samples imaged at lower magnification or if the micrograph should not contain the carbon edge and holey carbon films with larger spacing such as R1/4 200-mesh can be used to aid with aligning the stage to the area of interest and protecting areas from over-exposure in focusing and tracking routines.</li>
	<li>Place the slide on the platform in a glow discharge device. 
	NOTE: We use an in-house device in which an anode and platform has been machined into a vacuum desiccator and is powered by a high-frequency generator. After creating a vacuum, attach the high-frequency generator probe to the anode and power on the probe for 1 min to glow discharge the grid. The time necessary to glow discharge the grid can range from a few seconds to a minute. Varying the glow discharge time can be used to diagnose problems with specimen concentration and grids that appear dry with no vitreous ice.</li>
	<li>Remove the grid with a set of forceps, and lock the forceps closed with an elastic band.</li>
	<li>Add 100 &#956;l of 10 nm colloidal gold solution to the microcentrifuge tube with the minicells prepared in 1.4 and mix by gently flicking the tube with a finger. With a new pipette place 4 &#956;l of the mixture on the grid prepared in 2.2. 
	NOTE: Colloidal gold is available in a variety of sizes and care should be taken that the size of the gold is greater than 5 pixels given the pixel size of the micrographs to be processed at the acquired magnification, while not being too large to obscure features of interest.</li>
	<li>Prepare the plunge-freeze apparatus; fill the outer freezing container with liquid nitrogen and then fill the inner chamber with liquid ethane. Attach the forceps with the grid to the plunger rod and lock the plunger rod into the raised position. 
	NOTE: See Iancu et al.20 for a protocol describing the use of a commercial plunge-freeze apparatus.</li>
	<li>Blot the grid by carefully touching a piece of filter paper to the drop of sample until the meniscus between the grid and filter paper separates and the wicking on the filter paper stops, then immediately release the plunger rod, freezing the grid. Carefully remove the forceps from the plunger rod and place the grid into a grid holder.</li>
	<li>Prepare the cryo-EM transfer station by filling the loading area and absorption pump container with liquid nitrogen. Once the loading area is at liquid nitrogen temperature place the grid holder and a microscope specimen cartridge in the loading area.</li>
	<li>Carefully remove the lock ring, which is either a small threaded lock ring on earlier Polara microscopes or a C-style clip ring on later models; place the EM grid into the cartridge using forceps and then gently reattach the lock ring back onto the cartridge securing the grid.</li>
	<li>Remove the multiple specimen holder from the microscope and attach it to the transfer station. Place the specimen cartridge into the holder using cartridge forceps and retract the holder from the loading area and transfer the multiple specimen holder back to the microscope. 
	NOTE: See Chen et al.21 for a visual protocol detailing 2.1-2.9.</li>
</ol>
3. High-throughput Automated Tilt-series Collection
<ol>
	<li>Collection of Low-magnification Maps
	<ol>
		<li>Open a new Navigator window by clicking &#39;Open&#39; in the &#39;Navigator&#39; menu of SerialEM10 (http://bio3d.colorado.edu/SerialEM)</li>
		<li>Find grid squares that contain acceptable imaging conditions (i.e.,&#160;thin ice, no contamination, subject of interest) using the fluorescent screen at low-magnification (~2,300X for the minicell specimen). 
		NOTE: [Optional: This step can be automated with SerialEM by montaging the entire grid, however it can be faster to simply select a few areas manually.]</li>
		<li>Adjust the stage to eucentric height by tilting the specimen holder to 50&#176; then adjust the z-height until the x-y translation of the stage is minimal between the tilted and untilted views.</li>
		<li>Move to the center of the grid square and click the &#39;Add Stage Pos&#39; button in the Navigator window to store the current stage position.</li>
		<li>Continue steps 3.1.1-4 above until all acceptable grid squares stage positions have been saved.</li>
		<li>Open a new montage MRC file by clicking &#39;New montage&#39; in the &#39;File&#39; menu. In the Montage Setup Dialog that opens up, select a number of pieces in X and Y that will acquire the entire grid square (e.g.,&#160;10 x 10 for a standard 200 mesh grid). Use a high binning such as 8 and select the &#39;Move Stage Instead of Shifting Image&#39; and &#39;Skip correlations used to align pieces&#39; radio buttons.</li>
		<li>In the Navigator window click the first stage position and set it to be acquired by checking the &#39;Acquire&#39; checkbox. Repeat this for each stage position in the Navigator window.</li>
		<li>Open the Navigator Acquire Dialog by clicking &#39;Acquire at Points&#39; in the &#39;Navigator&#39; menu. Check the &#39;Acquire map image&#39; and &#39;Rough eucentricity&#39; checkbox and make sure that all other checkboxes are unchecked. Click &#39;Proceed&#39; to collect a montage at each stage position.</li>
	</ol>
	</li>
	<li>Tilt-series Acquisition
	<ol>
		<li>In the Navigator window select one of the acquired maps and click the &#39;Load map&#39; button.</li>
		<li>In the Navigator window click the &#39;Add Points&#39; button and select points in the map at which to acquire a tilt-series. Then click the &#39;Stop Adding Points&#39; button. Repeat for each map collected.</li>
		<li>In the Camera menu select &#39;Parameters&#39; and define the parameters for the Focus, Trial, and Record modes. [Optional: Dose-fractionated data can be specified in parameters for Record mode.]</li>
		<li>Select a point in the Navigator window and check the &#39;Tilt-Series&#39; check box. In the Tilt-Series Setup dialog window that opens select the parameters desired for the tilt-series collection. Repeat for the rest of the selected points in the Navigator window, but do not select the maps.</li>
		<li>In the Navigator menu again select the &#39;Acquire at Points&#39;. In the Navigator Acquire dialog choose &#39;Realign to item&#39;, Autofocus&#39; and &#39;Rough eucentricity&#39; as Preliminary tasks, and select &#39;Acquire tilt series&#39; as the Primary task, and select &#39;Close column valves at end&#39; to close the column when all of the points have been collected. Upon proceeding a tilt-series will be collected at each point in each map.</li>
	</ol>
	</li>
</ol>
4. High-throughput Automated Tilt-series Processing and Reconstruction Using Tomoauto
<ol>
	<li>Correction of Beam-induced Motion in Dose-fractionated data [optional] 
	NOTE: Tomoauto uses MOTIONCORR22 (http://cryoem.ucsf.edu/software/driftcorr.html) to remove beam-induced motion from dose-fractionated micrographs. NOTIONCORR16 must be installed on the system.
	<ol>
		<li>With the original tilt-series, the output log from SerialEM10 and the individual dose-fractionated images all in the current working directory, in a terminal execute the command: 
		&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; dose_fractioned_to_stack &#60;filename.st&#62; 
		&#60;filename.st&#62; is the name of the tilt-series to process.</li>
	</ol>
	</li>
	<li>Alignment and Reconstruction of Tilt-series 
	NOTE: Tomoauto by default uses IMOD13 (http://bio3d.colorado.edu/imod/) to handle tilt-series automated fiducial model generation, alignment, determination of the contrast transfer function (CTF), CTF-correction23, and reconstruction. Alternatively users have the option in tomoauto to use RAPTOR24 (included in IMOD) for automated fiducial model generation, CTFFIND425 (http://grigoriefflab.janelia.org/ctf) to determine the CTF, and tomo3d26 (https://sites.google.com/site/3demimageprocessing/tomo3d) for reconstruction or in any combination of software packages by configuration.&#160; This configuration as well as the parameters available in each package is handled by a global configuration file that can be edited to suit the values most commonly used in a lab while local configuration files can also be created to detail parameters used for a specific specimen, collection set or individual tilt-series. All packages that wish to be used must be installed on the system.
	<ol>
		<li>With the tilt-series in the current working directory, in a terminal execute the command 
		&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; tomoauto --CTF --mode=align &#60;filename.st&#62; &#60;fid_diam&#62; 
		&#60;filename.st&#62; is the tilt-series to be processed and &#60;fid_diam&#62; is the diameter of the fiducial markers in nanometers. This command will align and estimate the CTF of the tilt-series automatically. It is possible to skip CTF processing by removing the --CTF option from the command.</li>
		<li>Inspect the aligned tilt-series visually for any glaring errors in the alignment processing and inspect the estimated CTF by executing the commands: 
		&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 3dmod &#60;filename&#62;.ali 
		&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; submfg &#60;filename&#62;_ctfplotter.com 
		respectively, where &#60;filename&#62; is the name of the tilt-series without suffix. Also check the output of the tomoauto command to see the mean residual error generated by the alignment, which is a quantitative statistic of alignment quality.</li>
		<li>Given an acceptable alignment proceed processing by executing the command: 
		&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; tomoauto --CTF --mode=reconstruct &#60;filename.st&#62; &#60;fid_diam&#62; 
		with the same user substitutions as in 4.2.1. This command will correct the CTF, erase the fiducial markers from the tilt-series and compute the reconstruction. Again CTF processing can be skipped as in 4.2.1.</li>
		<li>[optional] To skip the visual inspection step and fully automate processing and reconstruction execute the command 
		&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; tomoauto --CTF &#60;filename.st&#62; &#60;fid_diam&#62;</li>
		<li>[optional] To use a specific local configuration, refer to the tomoauto documentation on how to generate a local configuration file, and then execute the command 
		&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; tomoauto [options]-L &#60;local_config&#62; &#60;filename.st&#62; &#60;fid_diam&#62; 
		where &#60;local_config&#62; is the name of the local configuration file.</li>
	</ol>
	</li>
</ol>
5. Sub-tomogram Averaging
NOTE: We use the i3 package15 (http://www.electrontomography.org/) to process sub-tomogram averaging experiments, however the protocol described applies generally to most available sub-tomogram averaging software packages16to process sub-tomogram averaging experiments, however the protocol described applies generally to most available sub-tomogram averaging software packages16-18.
<ol>
	<li>With the reconstructed tomogram in the current working directory open up the tomogram for particle picking by executing the command: 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; tomopick &#60;filename&#62;.rec 
	where &#60;filename&#62; is as in 4.2.2. In the window that opens use the left mouse button to click on first the basal body and then the needle tip to select an injectisome and use the up and down arrow-keys to riff through the slices of the tomogram. Select all visible injectisomes in this manner. This stores the coordinates in a text file defining the long axis of the structure as well as estimating two of the three Euler angles describing the orientation of the structure.</li>
	<li>Computationally extract 4003 voxel cubes from the tomogram centered at the midpoint of the defined long axis by executing the command: 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; clip resize -cx &#60;x&#62; -cy &#60;y&#62; -cz &#60;z&#62; -ix 400 -iy 400 -iz 400 \ 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#60;filename&#62;.rec &#60;filename&#62;_001.mrc 
	where &#60;x&#62;, &#60;y&#62;, &#60;z&#62; are the midpoint coordinates of the structure and &#60;filename&#62; is as in 4.2.2. The size of the extracted cube should vary by the structure and the magnification used, and should be large enough to adequately enclose the structure of interest, which for this sample is 4003 voxels.</li>
	<li>Down-sample (bin) the sub-tomogram by a factor of four to reduce the computation time for initial alignment by executing the command: 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; binvol -b 4 &#60;filename&#62;_001.mrc &#60;filename&#62;_001.bin4.mrc 
	where &#60;filename&#62; is as in 5.2.</li>
	<li>Apply the determined Euler angles to sub-tomograms and compute the global average to produce the initial template by executing the command. 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; I3totsum.sh</li>
	<li>Align and classify down-sampled sub-tomograms using a binary classification mask of the cytoplasmic area. Perform sub-tomogram averaging in Fourier space to minimize the missing wedge artifacts characteristic of tomography. For binning 4 data, please use SAMPFACT=&#34;4 4 4&#34;. 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; i3mramsacls.sh</li>
	<li>Repeat step 5.5 using sub-tomograms down-sampled by a factor of two (SAMPFACT=&#34;2 2 2&#34;) and once more with the original data (SAMPFACT=&#34;1 1 1&#34;). 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; i3mramsacls.sh</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomo3D+2.0+&#8211;+Exploitation+of+Advanced+Vector+eXtensions+(AVX)+for+3D+reconstruction.">Tomo3D 2.0 &#8211; Exploitation of Advanced Vector eXtensions (AVX) for 3D reconstruction.</a> J. Struct. Biol. 189 (2), 147-152 (2015).</li><li>Zhao, X., Zhang, K., Boquoi, T., Hu, B., Motaleb, M. A., Miller, K., James, M., Charon, N. W., Manon, M. D., Norris, S. J., Li, C., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-Electron+Tomography+Reveals+the+Sequential+Assembly+of+Bacterial+Flagella+in+Borrelia+burgdorferi.">Cryo-Electron Tomography Reveals the Sequential Assembly of Bacterial Flagella in Borrelia burgdorferi.</a> Proc Natl Acad Sci U S A. 110 (35), 14390-14395 (2013).</li><li>Hu, B., Margolin, W., Molineux, I. J., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bacteriophage+T7+Virion+Undergoes+Extensive+Structural+Remodeling+during+infection.">The Bacteriophage T7 Virion Undergoes Extensive Structural Remodeling during infection.</a> Science. 339 (6119), 576-579 (2013).</li><li>Liu, J., Hu, B., Morado, D. R., Jani, S., Manson, M. D., Margolin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=W:+Molecular+architecture+of+chemoreceptor+arrays+revealed+by+cryoelectron+tomography+of+Escherichia+coli+minicells.">W: Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells.</a> Proc Natl Acad Sci USA. 109 (23), e1481-e1488 (2012).</li><li>Russo, C. J., Passmore, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy:+Ultrastable+gold+substrates+for+electron+cryomicroscopy.">Electron microscopy: Ultrastable gold substrates for electron cryomicroscopy.</a> Science. 346 (6215), 1377-1380 (2014).</li></ol>

The authors declare that they have no competing financial interests.

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.

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

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

Research

JoVE Journal

Bioengineering

10.8K Views.  University of Texas Health Science Center at Houston. 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.

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

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Immunology and Infection

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

Biology

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

Biochemistry

User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for the high-resolution structure determination of biological macromolecules. Despite the remarkable advances in cryo-EM, there is still scope for improvement in various aspects of the single-particle analysis workflow. Single-particle analysis demands a suitable software package for high-throughput automatic data acquisition. Several automatic data acquisition software packages were developed for automatic imaging for single-particle cryo-EM in the last eight years. This paper presents an application of a fully automated image acquisition pipeline for vitrified biomolecules under low-dose conditions. 
It demonstrates a software package, which can collect cryo-EM data fully, automatically, and precisely. Additionally, various microscopic parameters are easily controlled by this software package. This protocol demonstrates the potential of this software package in automated imaging of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein with a 200 keV cryo-electron microscope equipped with a direct electron detector (DED). Around 3,000 cryo-EM movie images were acquired in a single session (48 h) of data collection, yielding an atomic-resolution structure of the spike protein of SARS-CoV-2. Furthermore, this structural study indicates that the spike protein adopts two major conformations, 1-RBD (receptor-binding domain) up open and all RBD down closed conformations.

Single-particle cryo-electron microscopy demands a suitable software package and user-friendly pipeline for high-throughput automatic data acquisition. Here, we present the application of a fully automated image acquisition software package, Latitude-S, and a practical pipeline for data collection of vitrified biomolecules under low-dose conditions.

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current ...

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...