Name: cryo-electron tomography remote data collection and subtomogram averaging
Uploaded: 2022-07-12T14:00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging at JoVE.com

We acknowledge Diamond Light Source for access to and support of the cryo-EM facilities at the UK&#39;s national Electron Bio-Imaging Centre (eBIC), funded by the Wellcome Trust, MRC, and BBRSC. We would also like to thank Andrew Howe for the acquisition of the Apoferritin tomogram (Movie 1), Ishika Kumar for the preparation and acquisition of the neuron tomogram (Movie 2), and Craig MacGregor-Chatwin for the Cyanobacteria lamella-tomogram (Movie 3).

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The authors have no conflicts of interest.

Tomo5 
The workflow description of the tomography software highlights one potential and most streamlined way for a (remote) batch tomography session setup. While the software is easy for beginners, some initial cryo-EM experience and basic tomography understanding can help with the setup. Critical steps are highlighted in the protocol and should help troubleshoot even if a different setup approach has been used. The advancement of the software will ease (remote) data collection and make cryo-ET more accessible to a wide user base.&#160;A few tips and tricks that can help troubleshoot commonly encountered problems are described below.
One important point to discuss is the choice of grids because, when tilting the specimen to &#177;60&#176;, grid bars at high tilts can obscure the view (Figure 8). On a TEM grid, the mesh size refers to the number of grid squares per unit length of the grid. Larger mesh numbers have more grid squares per unit length, a higher density of grid squares, and smaller grid squares, i.e., a 400-mesh grid has smaller squares than a 200-mesh grid. A good choice of grids for tomography is 200-mesh or 300-mesh grids. As shown in Figure 8, the available area to collect is reduced as the grid is tilted. At &#177;60&#176; tilt, a 300-mesh grid will have a small field of view on which a full tomogram can be acquired. The advantages of 200-mesh grids are that the larger grid squares make molecular tomography setup faster, and with the increased grid-square area, one square will likely be enough for an overnight collection. The disadvantage is that 200-mesh grids are more fragile, so handling and clipping require more finesse.
Moreover, if using holey support film (see Table of Materials) on EM grids, the hole spacing must be considered for the setup of the focus and tracking region in relation to the exposure region. Ideally, the beam diameter at the desired magnification should be small enough to cover the carbon area adjacent to the exposure area along the tilt axis for optimal and fast setup. This way, potential regions of interest in each hole can be acquired.
As the software&#39;s eucentric height routine is currently not as robust, such as the serialEM routine, the following tips can work around that problem. If the eucentric height determination fails using the eucentric height preset, one can use the overview preset instead and re-run &#34;Auto-eucentric by stage tilt&#34;; this can solve issues if the eucentric height is far away from 0. If this succeeds, one can re-run &#34;Auto-eucentric by stage tilt&#34; with &#34;Eucentric Height&#34; presets to improve precision. If it fails, one can run &#34;Auto-Eucentric by beam tilt&#34; with the eucentric height preset and then re-run &#34;Auto-Eucentric by stage tilt&#34; or manually set the z-height consolidated by &#34;Auto-Eucentric by beam tilt&#34; in the TEM User Interface under &#34;Stage&#34; settings. In case grids with a repeating pattern of holes are used, they may prevent the identification of a single cross-correlation peak. One can try altering the eucentric height preset to a lower defocus offset such as &#8722;25 &#181;m and/or a shorter exposure time to reduce cross-correlation from the hole patterns. On the other hand, using lacey grids/lamella may not provide sufficient signal for a strong cross-correlation peak. One can try altering the eucentric height preset to a greater defocus offset such as &#8722;75 &#181;m and/or an extended exposure time to enhance the cross-correlation peak. Another option is to adjust image filter settings; they can be found in the &#34;Preparation&#34; tab. Options to adjust the filter settings can be set for low (Overview/Gridsquare), medium (Eucentric Height), and high magnification (Tracking/Focus) to find the optimal cross-correlation peak for each preset. The required input is one image, i.e., at 0&#176; and one at 5&#176;, followed by clicking Compare to compare both images. Recommended starting value for the longest wavelength is one-quarter of the scale bar in the image and for the shortest wavelength is one-fortieth of the scale bar. If the peak is not robustly identified, one can optimize&#160;the settings until a convincing peak can be found. There is no need to re-acquire images every time; simply pressing &#34;Compare&#34; is enough. If TOMO still fails to automatically find the eucentric height, the manual eucentric height calibration can be used. One should center over a reasonably large ice crystal in overview magnification in the &#34;Preparation&#34; tab, then go to the the &#34;Stage control&#34; of the TEM User Interface, set alpha to &#8722;30&#176;, and adjust the stage z-value to re-center the crystal using the fluorescent screen image. Selecting &#34;High Resolution&#34; and &#34;High Contrast&#34; settings in the TEM User Interface will make this simple (buttons at the bottom of the fluorescent screen window). Optionally, if there is access to a camera with live mode, then this can be used to determine the eucentric height; it will be easier than on the fluorescent screen.
The biggest limitations in Tomo5 versions prior to 5.8 are the missing medium magnification montages, missing dose symmetric scheme, and problems related to eucentric height finding. These exist in serialEM, a freeware with rapid development and community support, a robust eucentric height routine, and the option to script, i.e., a custom-built dose symmetric scheme. From version 5.8 onwards in Tomo5, the most commonly encountered problem for finding the eucentric height, i.e., an unsuccessful looping around the target z-value, has been solved by implementing the option to set a eucentric height acceptance criterion. However, with different grid and sample types, it is highly recommended to adjust the image filter settings to reflect the unique imaging conditions of individual sessions and to give the best possible cross-correlation peak to find the eucentric height and for the focus and tracking region to work reliably during tomogram acquisition.

Overall, many facilities have rapidly adapted to remote operation during the pandemic. The Tomo5 software provides an easy access and user-friendly route to tomography which is well suited for remote operation. The advances made in the software will no doubt continue to make remote data collections and tomography collection in general more mainstream in the community.
emClarity 
As emClarity uses a template-based particle picking method, it needs a template for the object of interest. The particle picking (step 2.6) is very sensitive and key to the final structure. Before averaging and alignment (step 2.9), one must ensure to check carefully and manually remove the false positives. When a template is not available, emClarity might not be easy to use, but it is possible to use other software, for example, Dynamo37 and PEET48, to create an initial model.
For heterogeneous samples, emClarity is equipped with a classi&#64257;cation method that enables users to focus on specific features with different scales. It is helpful to run a few cycles of alignments before classification and run it at a higher binning (such as bin 4 or bin 3).
The up-to-date version of the software (V1.5.3.11) has significant updates compared with the first release (V1.0)17. These include, but are not limited to, a handedness check during CTF estimation (step 2.3); symmetry for alignments (CX, I, I2, O); calculation of per-particle 3D sampling functions (3DSF); a switch to MATLAB 2019a for compatibility and stability; and reconstruction using the raw projection images (cisTEM). The software will continue to improve for various samples, and the newest announcements can be found online (see Table of Materials).

Cryogenic electron microscopy (cryo-EM) is widely known to have experienced a renaissance period, accelerating it to become a core and centrally useful tool in structural biology. The development and utilization of direct electron detectors1,2,3, improved microscopes and electron sources3,4,5, improvements in automation/throughput6,7,8,9, and computational advances in single-particle analysis10,11,12,13,14 and tomography15,16,17 are all, in part, responsible for the recent success of the technique. These technological drivers have developed cryo-EM&#39;s capability to solve biological macromolecular structures under cryogenic and native conditions. The resolutions that are readily obtainable are sufficient for atomically accurate modeling and have brought the technique to the forefront of the structural biology arena. A reductionist approach to expressing and purifying a biological target of interest has long proven successful in macromolecular crystallography (MX) for basic biological research, drug discovery, and translational science. In the same approach, cryo-EM can now deliver results that parallel high-resolution MX studies. The current major success in the cryo-EM branch of structural biology is called single particle analysis (SPA), which acquires 2D projection images typically of a purified protein specimen18 to obtain thousands of views of a biological macromolecule19. These images (1) contain information from a range of views that fully represent the orientations of the target in 3D space and (2) capture the object conformational heterogeneity, which can later be separated and investigated.
An alternative approach to acquiring these 2D projection images of biological samples, even in situ and without purification, is cryo-electron tomography (cryo-ET). Cryo-ET takes a series of images of the same object at tilted angles by mechanically rotating the specimen. Thus, the 2D projections collected in SPA, representing the angular poses of the molecule of interest, are inherently collected as part of the cryo-ET imaging experiment20. Tomographic tilt series are then reconstructed into a tomogram that contains 3D representations of the imaged macromolecular complexes. The nature of tomographic data collection does, to a degree, decrease the reliance on averaging to achieve a full 3D representation of a molecule from a collection of 2D images. However, due to current stage designs, the specimen is typically tilted from &#8722;60&#176; to +60&#176;, leaving a missing wedge21 of information in the tomographic 3D reconstruction.
The 3D reconstructions in a single tomogram then have a missing wedge of information and low signal to noise. Individual macromolecules may be extracted as subtomograms and averaged together to tackle this. Where each macromolecule in a subtomogram is found at a different orientation, the missing wedge is oriented differently in each subtomogram of the target object, so averaging over many copies fills in information due to the missing wedge. Recent developments in image processing have also attempted to train artificial intelligence neural networks to fill in the missing wedge with meaningful data22. This averaging process also increases the signal to noise, akin to the goal of averaging in single particle analysis, so the reconstruction&#39;s quality and resolution improve. If the molecule of interest possesses symmetry, that too may be defined and employed during averaging, further improving reconstruction resolution. The extraction of 3D volumes of a macromolecule from a tomogram into a set of subtomograms and their subsequent processing is known as subtomogram averaging (STA)23. Where each subtomogram represents a unique copy of the molecule being studied, any structural heterogeneity may be interrogated using the STA workflow. As commonly utilized in the SPA workflow, classification techniques may be employed during STA to dissect the conformational states of the complex of interest. As well as STA enabling high-resolution reconstruction in cryo-ET, this approach makes the technique a powerful tool to interrogate the structural mechanisms of macromolecules in their native cellular environment or of targets often not amenable to SPA24,25,26.
Electron tomography has a long history of determining the 3D ultrastructure of cellular specimens at room temperature27. The acquisition of views by physical tilting of the specimen provides enough information for the 3D reconstruction of an object at cellular-length scales and is particularly important when cellular structures lack the regularity for averaging. Cells may also be frozen onto substrates for cryo-ET imaging at the cell edges where the specimen is thin enough to be electron transparent. Under these conditions, STA may be employed to determine macromolecular structures in a cellular environment, albeit when the specimen is thin enough to be electron transparent28. However, when combined with additional preparative techniques, including cryo-correlative light and electron microscopy (cryo-CLEM) and focused ion beam milling (cryo-FIB), cryo-ET can be used to image inside whole cells under cryogenic conditions29. This brings together the power of cryo-ET to study the cellular ultrastructure with the power of STA to determine the structures of macromolecular complexes in situ while identifying their cellular location30 and providing snapshots of complexes engaged in dynamic processes31. The ability of the technique to image cellular specimens and employ STA in several studies has highlighted the power of the technique to solve macromolecular structures in situ, even at resolutions comparable to SPA32. A further benefit is found in the knowledge of the original location of the macromolecule, represented by the final classified 3D reconstruction in the tomogram30. Therefore, the macromolecular structure can be correlated with the cellular ultrastructure. These observations across length scales will presumably lead to important findings where structural mechanisms may be correlated with cellular changes in the context of functional studies.
Cryo-ET and STA allow data collection in three major workflows: molecular, cellular, and lamella tomography. The structures of purified macromolecular complexes may be determined by cryo-ET by molecular tomography. Determining protein structures in their cellular environment where the cell is thin enough may be described as cellular tomography. More recently, with the development of cryogenic targeting and milling, these same techniques may be applied in lamella tomography workflows to determine the protein structures deep inside the cell in their native environment while revealing the cellular context in which those proteins are observed. Different data collection strategies can be used depending on the available software packages and, most importantly, depending on the requirement of the specimen. Molecular or non-adherent samples on a copper TEM grid of a purified protein typically require less handling and, thus, remain flat and undamaged in ideal cases. Electron tomograms can easily be set up in series across a holey-carbon grid to quickly acquire tens to hundreds of tomograms in a systematic manner. The simplest way for users to set up molecular tomography samples where proteins are abundantly present on the grid would be to use Tomo5 (software for 3D electron tomography used in the present study, see Table of Materials). Other tomography software such as Leginon9 and serialEM6 are also available; they offer more setup options for more personalized approaches for data collection but are more complex and consequently can be harder to navigate, particularly for users new to tomography and users accessing their session remotely. For a facility with a large and diverse user base, Tomo5 is easy to operate in a remote environment and to train users in. For adherent cells, grids typically require more handling steps, and the necessity to use fragile gold grids increases the need for improved care in handling and data collection strategies. To facilitate finding a cellular region of interest and avoid occlusion from the grid itself at high tilt angles, it is also beneficial to use larger mesh sizes, but at the cost that they are inherently more fragile. For lamella samples, the fragility of the sample is determined by the quality of the lamella, which can be variable. These factors increase the setup time and considerations, but the increased adaptability and robustness again make Tomo5 suitable for this type of data collection. However, specialized data collection scenarios exist for each workflow. BISECT and PACE-tomo (both run in&#160;SerialEM) introduces the possibility of scripted beam-image shifting during tomography acquisition to increase tomogram collection speed28, particularly in molecular tomography. Medium magnification montages (MMM) in SerialEM6,7,33 can better identify and precisely target molecular features in all workflows, although, at the time of writing, these features are beginning to be implemented in Tomo5.
Like SPA, cryo-ET and STA are becoming increasingly accessible through the improvements made to acquisition software and a wealth of available packages for subtomogram averaging16,17,32,34,35,36,37,38. In addition, during the pandemic, enabling remote access to cryo-EM instrumentation became essential to the continued operation of national facilities like the electron Bio-Imaging Centre (eBIC) at Diamond Light Source (DLS), UK. These developments have made cryo-ET more accessible and robust for researchers wishing to utilize the technique. Once data have been acquired, STA is an essential tool for analyzing recurrent objects to obtain maximum resolution reconstruction and allow the classification of macromolecular heterogeneity. The current protocol aims to provide a detailed walk-through of preparing a cryo-TEM microscope for cryo-ET data collection and how to perform subtomogram averaging using emClarity on a molecular tomography dataset of apoferritin as an example. The use of emClarity (software for high-resolution cryo-electron tomography and subtomogram averaging, see Table of Materials) requires running scripts from the command line, so a level of familiarity with Linux/UNIX systems is assumed.
The remote connection depends on the network environment in each institute/facility. At eBIC, the remote system uses programs&#160;that allow remote data collection on the specific network configuration used at Diamond. Remote connection to the microscope is facilitated by two platforms: NoMachine and TeamViewer (see Table of Materials). Using the program NoMachine, the user may log onto a remote Windows desktop. The remote Windows desktop provided by NoMachine resides on the same network as the microscope and, thus, acts as a virtual support PC to the microscope. From the virtual support PC, the user connects to the microscope via TeamViewer providing direct access and control to the microscope PC running TUI and Tomo.
The present protocol consists of two parts (step 1 and step 2). Step 1 focuses on remote cryo-ET data acquisition using Tomo5 (software for 3D electron tomography). The walk-through for a (remote) session captures images at increasingly higher magnifications to ultimately allow the user to direct the tomography software to target specimen areas for tomographic data collection. Figure 1 summarizes this process. Step 2 details cryo-ET STA data processing using emClarity (software for high-resolution cryo-electron tomography and subtomogram averaging). Figure 9 summarizes this process.
The protocol is intended for a remote audience. It assumes the person physically at the microscope and loading the samples has done the direct alignments and taken care of the camera tuning and gain reference acquisition. For this protocol, a three-condenser lens system with an autoloader is assumed. For further detailed guidelines on the tomography software, a detailed manual by the manufacturer is available in the Windows Start button where the software was loaded from.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tomography</td>
			<td>Thermo Fisher Scientific</td>
			<td>5.9.0</td>
			<td>Internal terminology: Tomo5 in document</td>
		</tr>
		<tr>
			<td>TEM server</td>
			<td>Thermo Fisher Scientific</td>
			<td>7.10.1</td>
			<td></td>
		</tr>
		<tr>
			<td>TIA</td>
			<td>Thermo Fisher Scientific</td>
			<td>5.10.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DigitalMicrograph</td>
			<td>Gatan</td>
			<td>3.44</td>
			<td></td>
		</tr>
		<tr>
			<td>emClarity</td>
			<td>Open-Source software</td>
			<td>1.5.3.11</td>
			<td>Software for high-resolution cryo-electron tomography and subtomogram averaging</td>
		</tr>
		<tr>
			<td>IMOD</td>
			<td>Open-Source software</td>
			<td>4.11</td>
			<td>Modeling, display and image processing programs used for 3D reconstruction and modeling of microscopy images with a special emphasis on electron microscopy data</td>
		</tr>
		<tr>
			<td>MotionCor2</td>
			<td>Free for academic use</td>
			<td>1.1.0</td>
			<td>A multi-GPU program that corrects beam-induced sample motion recorded 
			on dose fractionated movie stacks</td>
		</tr>
		<tr>
			<td>ETomo</td>
			<td>Open-Source software</td>
			<td>4.11</td>
			<td>ETomo is an interface for running a subset of IMOD and PEET commands.&#160;</td>
		</tr>
		<tr>
			<td>NoMachine</td>
			<td>NoMachine, freeware</td>
			<td>7.9.2</td>
			<td>Remote desktop software</td>
		</tr>
		<tr>
			<td>TeamViewer</td>
			<td>TeamViewer AG</td>
			<td>-</td>
			<td>Remote access and remote control computer software</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quantifoil (holey support film) EM grids</td>
			<td>Quantifoil</td>
			<td>-</td>
			<td>A flat film of carbon with pre-defined hole size, shape and arrangement</td>
		</tr>
		<tr>
			<td>Instrumentation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titan Krios microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td>Titan Krios G2</td>
			<td></td>
		</tr>
		<tr>
			<td>K3 camera and GIB energy filter</td>
			<td>Gatan</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 4 camera and Selectris X energy filter</td>
			<td>Thermo Fisher Scientific</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Website</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Website 1: https://github.com/bHimes/emClarity/</td>
			<td>-</td>
			<td>-</td>
			<td>Link to download the emClarity software package</td>
		</tr>
		<tr>
			<td>Website 2: https://bio3d.colorado.edu/imod/</td>
			<td>-</td>
			<td>-</td>
			<td>Link to download IMOD</td>
		</tr>
		<tr>
			<td>Website 3: https://github.com/ffyr2w/emClarity-tutorial</td>
			<td>-</td>
			<td>-</td>
			<td>Link to the emClarity online tutorial</td>
		</tr>
		<tr>
			<td>Website 4: https://emcore.ucsf.edu/ucsf-software</td>
			<td>-</td>
			<td>-</td>
			<td>Link to download MotionCor2</td>
		</tr>
		<tr>
			<td>Website 5: https://github-wiki-see.page/m/bHimes/emClarity/wiki</td>
			<td>-</td>
			<td>-</td>
			<td>Link to the newest announcements including updates and bug fixs for emClarity</td>
		</tr>
	</tbody>
</table>

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.

For cellular and lamella samples, the data collection strategy largely depends on the sample and the goal of the imaging study (Figure 1). The targeting approach depends on whether the molecular target is in situ or prepared from the purified macromolecular complex for high-resolution reconstruction specimens containing molecular targets. For purified complexes vitrified onto holey (carbon) grids, targeting can simply be based on imaging in the holes of the (carbon) support film. For in situ work, the targeting approach requires knowledge of the location of the molecular entity based on correlative data or known low-magnification cellular landmarks. Cellular landmarks would ideally be identifiable when taking overview images and, if sufficient to roughly localize regions of interest, can provide a quick way to confirm target identity with a search image. However, if the observable events are rare, then medium magnification search images may be needed to qualify the target is correct. Search maps are medium magnification montages of search images and can, thus, make target finding much easier, where they can be acquired at a magnification at which the feature of interest is visible. Search maps can then be screened to find and set up target batch positions. For specimens containing cellular features for ultrastructural reconstruction, the targeting approach is similar, though equally dependent on the visibility of the cellular event at various magnifications and its prevalence in the sample.
The data acquisition strategy must also be considered; in all cases, the study&#39;s goal largely determines how the data is collected. For reconstruction of the cellular ultrastructure, a low magnification (20-5 &#197;/px) and large field of view may be appropriate, but high magnifications are required to reconstruct molecular or high-resolution detail (5-1 &#197;/px). A dataset collected at 1.5 &#197;/px under ideal conditions will only physically be able to produce a reconstruction at the Nyquist frequency of 3.0 &#197;/px; however, in reality, many factors, including but not limited to specimen thickness, size, and heterogeneity, all affect the obtained reconstruction quality. The exact imaging parameters also balance the magnification based on the study&#39;s goal with the field of view to contain enough information. This article presents a subtomogram averaging case reaching 2.86 &#197;, but additional studies17,42,43,44,45 are presented in Table 1 to illustrate the collection parameters associated with studies targeting different outcomes17,42,45,46.
Once a targeting workflow and data acquisition regime for cryo-ET is established, data collection of many different sample types is possible. Representative tomograms of a variety of specimens are presented here: molecular samples such as apoferritin (Movie 1), thin cellular processes (Movie 2), and FIB milled lamella of the thick cellular specimen (Movie 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63923/63923fig01.jpg" /> 
Figure 1: Overview of the tomography workflow setup. The cryo-ET imaging workflow described in the protocol is shown as a flow chart. The images expected to be acquired are shown for the cellular and molecular workflow. The presented naming follows the Tomo5 convention, although most tomography acquisition software shares common principles for collecting these images. <a href="https://www.jove.com/files/ftp_upload/63923/63923fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63923/63923fig02.jpg" /> 
Figure 2: Preparation tab and search preset conditions. (A) Overview image of the whole tab. Imaging condition presets are set in this tab, and the &#34;Calibrate Image Shift&#34; and &#34;Image Filter Settings&#34; can be found in the &#34;Tasks&#34; drop-down. (B) Zoom-in of the &#34;Presets&#34; drop-down where each preset can be selected for individual imaging conditions setup. (C) Image depicting an adequate magnification to fit both the exposure and the &#34;Focus and Tracking&#34; area into the field of view. <a href="https://www.jove.com/files/ftp_upload/63923/63923fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63923/63923fig03.jpg" /> 
Figure 3: Dose calculations. Example dose calculations for possible tomogram acquisition schemes where the dose rate has been measured over vacuum. The two calculations determine the exposure time (s) for each tilt, whether targeting an optimal dose per tilt or an optimal total dose for the full tilt series. In subtomogram averaging, it is common practice to target an optimal dose per tilted image in the range of 3.0-3.5 e-/&#197;2. In both cases, the &#34;Fractions (Nr.)&#34; are set to 6 to achieve ~0.5 e-/&#197;2 of dose per movie frame of the tilt for sufficient signal to perform motion correction. <a href="https://www.jove.com/files/ftp_upload/63923/63923fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63923/63923fig04.jpg" /> 
Figure 4: Atlas tab. Overview image of the &#34;Atlas&#34; tab. The image has been cropped to avoid empty grid position spaces. The &#34;Tasks&#34; menu contains session setup preferences and grid selection spaces for individual selection of all grids inventoried and an option to acquire a single atlas after the cassette has been removed from the autoloader. A selected grid can be reset and then re-acquired. <a href="https://www.jove.com/files/ftp_upload/63923/63923fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63923/63923fig05.jpg" /> 
Figure 5: Calibrated image shifts. Image depicts an exposure and search image. The red cross in the search image is the shifted marker to correct the offset between exposure and search. One should redo image shift calibrations at the session start or after changing the imaging condition presets. <a href="https://www.jove.com/files/ftp_upload/63923/63923fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63923/63923fig06.jpg" /> 
Figure 6: Auto functions and apertures. (A) The &#34;Auto Functions&#34; tab depicts the &#34;Presets&#34; drop-down and the &#34;Task&#34; selection. Underlined in blue is the &#34;Thon Ring&#34; preset required for &#34;Autostigmate&#34; (also underlined in blue) and &#34;Autocoma&#34;. One should select respective presets for each task and then press the Start button. (B) Apertures are found in the TEM User Interface. Select desired &#34;Objective aperture&#34; after the auto functions have been performed and run &#34;Autostigmate&#34; with the aperture in. <a href="https://www.jove.com/files/ftp_upload/63923/63923fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63923/63923fig07..jpg" /> 
Figure 7: Tomography tab overview. Images show the Tomo 5.8 user interface. (A) Batch positions. The latest functions depicted in the image: the &#34;Acquire Search Map&#34; option; tomogram positions are shown in atlas view with the zoom-in option; highlighted at the top right is &#34;Select Positions&#34;; below all four positions have been selected to &#34;Update Defocus&#34; parameters. (B) Three positions are selected on the search map, labeled 1, 2, and 3. Position 1 will not pose a problem when &#34;Refine All&#34; is run. However, if in a high target density setting such as when lamellae positions are selected, as depicted for position 2 and position 3, then &#34;Refine All&#34; will run the &#34;Tracking&#34; and &#34;Focus&#34; routine on the &#34;Exposure&#34; region of position 2, exposing the target before the tomogram is acquired. The grid type is R1.2/1.3. Scale bar = 1.2 &#181;m. (C) Data acquisition. Selected positions setup in &#34;Batch Positions&#34; can now be individually acquired. <a href="https://www.jove.com/files/ftp_upload/63923/63923fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63923/63923fig8v2.jpg" /> 
Figure 8: Defining the maximum tilt angle. Figure depicts a step-by-step approach to determining the maximum tilt range for tomogram acquisitions. (A) 0&#176;&#160;overview and a search map in the center of the grid square. To test the tilt range, the angle can be set in the tomography software with &#34;Set Tilt (&#176;)&#34;&#160;by typing the desired value and then pressing Set. (B) The stage has been tilted to &#8722;60&#176;, showing that a corner of the search map would not be fully acquired at &#8722;60&#176;. (C) The stage has been tilted to 60&#176;. By counting the holes that have disappeared at &#177;60&#176;, one can get an idea about the tilt range of a grid square. Scale bars = 2.5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63923/63923fig08v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63923/63923fig09.jpg" /> 
Figure 9: emClarity flowchart. The flowchart described the various steps for cryo sub-tomogram averaging. Scale bars = 50 nm. <a href="https://www.jove.com/files/ftp_upload/63923/63923fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/63923/63923fig10.jpg" /> 
Figure 10: Template matching using emClarity. (A) A typical micrograph of apoferritin on a graphene-coated grid. Defocus: &#8722;3.430 &#181;m. (B) A slice of the tomogram overlaid with model points after template searching. (C) A top and (D) a side projection views of model points, indicating a single flat layer of monodispersed apoferritin particles. Scale bars = 50 nm. <a href="https://www.jove.com/files/ftp_upload/63923/63923fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/63923/63923fig11.jpg" /> 
Figure 11: Cryo-ET STA of apoferritin. (A) The final map after 21 cycles of sub-tomogram alignment. (B) The final map&#39;s Fourier shell correlation (FSC) plot with a reported resolution of 2.86 &#197;, containing 38 cone FSCs. (C) Representative density maps (fitted with PDB model 6s6147). <a href="https://www.jove.com/files/ftp_upload/63923/63923fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:font-size="8px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Cryo-ET type</td>
			<td>&#197;/px</td>
			<td>Tilt range (+/-)</td>
			<td>Tilt step (&#176;)</td>
			<td>Defocus range (&#181;m)</td>
			<td>Total dose (e-/&#197;2)</td>
			<td>Resolution</td>
			<td>Raw data</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Apoferritin</td>
			<td>Purified/Molecular (STA)</td>
			<td>1.34</td>
			<td>60</td>
			<td>3</td>
			<td>1.5 &#8211; 3.5</td>
			<td>102</td>
			<td>2.86</td>
			<td>EMPIAR-10787</td>
			<td>17 &#38; this paper</td>
		</tr>
		<tr>
			<td>HIV-1 Gag</td>
			<td>Purified/Molecular (STA)</td>
			<td>1.35</td>
			<td>60</td>
			<td>3</td>
			<td>1.5 &#8211; 3.96</td>
			<td>120</td>
			<td>3.1</td>
			<td>EMPIAR-10164</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Ribosome</td>
			<td>Purified/Molecular (STA)</td>
			<td>2.1</td>
			<td>60</td>
			<td>3</td>
			<td>2.2 &#8211; 4.3</td>
			<td>120</td>
			<td>7</td>
			<td>EMPIAR-10304</td>
			<td>42</td>
		</tr>
		<tr>
			<td>SARS-CoV-2 spike</td>
			<td>Lamella/Molecular (STA)</td>
			<td>2.13</td>
			<td>54</td>
			<td>3</td>
			<td>2 &#8211; 7</td>
			<td>120</td>
			<td>16</td>
			<td>EMPIAR-10753</td>
			<td>45</td>
		</tr>
		<tr>
			<td>Neuron axon structure</td>
			<td>Cellular (ultrastructural)</td>
			<td>5.46</td>
			<td>60</td>
			<td>2</td>
			<td>3.5 &#8211; 5</td>
			<td>90</td>
			<td>N.D.</td>
			<td>EMPIAR-10922</td>
			<td>47</td>
		</tr>
	</tbody>
</table>
Table 1: Collection parameters for several cryo-ET studies. Studies targeting the reconstruction of molecular detail from purified or in situ proteins compared to a study aiming to resolve and segment the ultrastructural cellular features.
Movie 1: A tomogram of apoferritin samples on a normal EM grid and then imaged with a cryo-TEM equipped with an ultra-high-resolution camera with a compatible filter. Tilt series were acquired with a dose-symmetric scheme, with a tilt span of 54&#176; and a total dose of 134 e-/A2 in the electron tomography software. Scale bar = 50 nm. <a href="https://www.jove.com/files/ftp_upload/63923/movie1_ApoF_50nmscale.mp4" target="_blank">Please click here to download this Movie.</a>
Movie 2: A tomogram of a primary neuron grown on an EM grid and then directly imaged with a cryo-TEM equipped with an ultra-high-resolution camera with a compatible filter. The tilt series were acquired with a dose-symmetric scheme, with a tilt span of 60&#176; and a total dose of 120 e-/A2 in the electron tomography software. Scale bar = 100 nm. <a href="https://www.jove.com/files/ftp_upload/63923/Movie2_neuron_100nm.mp4" target="_blank">Please click here to download this Movie.</a>
Movie 3: A tomogram of a Cyanobacteria on an EM grid, subjected to FIB milling and then imaged with a cryo-TEM equipped with a high-speed camera and a compatible filter. The tilt series were acquired with a dose-symmetric scheme, with a tilt span of 50&#176; and a total dose of 120 e-/A2 in the electron tomography software. Scale bar = 87.2 nm. <a href="https://www.jove.com/files/ftp_upload/63923/Movie3_lamella_100pxScale.mp4" target="_blank">Please click here to download this Movie.</a>
Supplementary File 1: The parameter file template for estimating the defocus. <a href="https://www.jove.com/files/ftp_upload/63923/63923_Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Data collection and microscope setup details. <a href="https://www.jove.com/files/ftp_upload/63923/63923_Supplementary Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 2: List of commands in the order of execution. <a href="https://www.jove.com/files/ftp_upload/63923/63923_Supplementary Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging at JoVE.com

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

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 ...

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