We thank Bill Anderson, Charles Bowman, and Jean-Christophe Ducom (TSRI) for help with microscopy, Leginon installations, and data transfer infrastructure. We also thank Gordon Louie (Salk Institute) and Yong Zi Tan (National University of Singapore) for the critical reading of the manuscript. We thank Chris Russo (MRC Laboratory of Molecular Biology, Cambridge) for providing us with the plasmid for expression of DPS.&#160;This work was supported by grants from the US National Institutes of Health (U54AI150472,&#160;U54 AI170855, and R01AI136680 to DL), the National Science Foundation (NSF MCB-2048095 to DL), the Hearst Foundations (to DL), and Arthur and Julie Woodrow Chair (to J. P. N.). T.S. is supported by an F32 postdoctoral fellowship from the National Institutes of Health (GM148049).

1. Sample preparation
<ol>
	<li>Use grids containing gold foil and gold grid support16 (see Table of Materials) because tilted data collection can accentuate beam-induced motion17. 
	NOTE: For the present study, samples on grids were vitrified using the manual plunging and blotting technique18 in a humidified (greater than 80%) cold room (~4 &#176;C).</li>
	<li>Avoid using grids containing copper support and carbon foil or a continuous layer of amorphous carbon unless absolutely necessary, as these grids may lead to greater beam-induced motion16 when the specimen stage is tilted. 
	NOTE: Alternative support layers, such as graphene/graphene oxide, appear to reduce beam-induced movement compared to amorphous carbon19,20.</li>
	<li>Pre-screen grids and identify regions characterized by acceptable ice thickness and particle distribution. Grids containing too tightly packed particles will lead to particle overlap during tilted data collection, which may affect downstream data-processing steps. 
	NOTE: These steps are subjective since identifying good areas of ice is performed by visually inspecting defocused images for regions where particle contrast is clear. This may not be feasible for all samples since some samples will not distribute efficiently in areas of thin ice, leading to challenges during data collection (described in the Discussion section).</li>
	<li>Vitrify the grids containing your protein sample. Here, for demonstration purposes, we use DNA Protection during Starvation (DPS) protein (see Table of Materials) at a range from 0.1-0.5 mg/mL with gold foil and gold support grids. 
	&#8203;NOTE: The protein was purified as described previously, except no TEV protease cleavage was performed21. The protein concentration range for a sample of interest will have to be optimized individually, since it is hard to gauge an ideal range that is universally applicable and will almost certainly vary between different samples.</li>
</ol>
2. Setting up tilted data collection
<ol>
	<li>Align the microscope to ensure parallel illumination of the specimen and minimize coma aberrations22. 
	NOTE: The microscope must be well aligned for standard SPA data collection without stage tilt. No special alignments are necessary for tilted data collection, but a good alignment will ensure that targeting and imaging proceed smoothly. A general scheme comparing tilted and untilted data collection is provided in Figure 2.</li>
	<li>Record a grid atlas without stage tilt to identify squares suitable for data collection or manually inspect squares at the magnification used in Square Acquisition Node. Look for squares where the foil is intact, does not look dehydrated, and has ideal ice thickness. 
	NOTE: Square Acquisition Node is the low-magnification node used for multi-scale imaging in Leginon.
	<ol>
		<li>For typical untilted automated data collection, record a grid atlas, which provides an overview of the overall grid quality and an initial indication of suitable areas for data collection.</li>
		<li>Subsequently, select suitable squares through the atlas and submit them to the queue. Then, either through manual selection of holes or through the automated EM hole finder, queue and submit the hole targets.</li>
		<li>Finally, use the automated EM hole finder for submitting high magnification exposure targets. 
		NOTE: For tilted data collection, squares may need to be queued manually for consistent results, especially if the optimal tilt angle has not been pre-determined and is likely to be adjusted during data collection. The grid atlas could also be recorded using a pre-defined stage tilt if the tilt angle used for data collection had previously been established.</li>
	</ol>
	</li>
	<li>Move the specimen stage to a square of interest.</li>
	<li>Determine the eucentric height for the stage position using &#945;-wobbler at &#177; 15&#176; stage tilt. Adjust the Z-height to bring the stage to eucentric height using the keypad panel for the microscope. Ensure the image shift is minimal during the &#945;-wobble routine. 
	NOTE: If the eucentric height is not properly identified, a large image shift will be observed upon tilting the stage at the square magnification. This can also happen if there are local deformations on the grid, for example, if the grid is broken or severely bent in the vicinity of the imaged area. Although it is best to avoid such regions for data collection, it is imperative to accurately estimate eucentric height if these represent one of the few promising regions for data collection. Figure 3 shows how targeting without properly identifying eucentric height can cause large image shifts in the square magnification.</li>
	<li>Find a more accurate Z-height, use the Focuser node, and press Simulate.
	<ol>
		<li>Typically, estimate the Z-height in the Focuser node at the magnification used in Square Acquisition Node. 
		NOTE: The Focuser node focus sequence can also include a fine Z focus estimation at the Hole Acquisition node (a tool in Leginon software) magnification during data collection.</li>
		<li>Adjust the settings for the Focuser node and enable/disable the fine Z focus option during the initial queuing of squares. 
		NOTE: It is important to ensure that eucentric height is accurately identified when automated data collection begins, which may require re-enabling fine Z focus (step 2.10).</li>
	</ol>
	</li>
	<li>Tilt the specimen stage to the desired tilt angle for data collection at the true eucentric height, and re-center the stage if necessary. 0&#176;, 30&#176;, and 60&#176; tilt angles were used for this study. Press Simulate in the Square Acquisition node to begin queuing targets for Hole Acquisition node exposures. 
	NOTE: As indicated in step 2.2.1, the grid atlas can be recorded using a pre-defined stage tilt, which would obviate the need to tilt the stage again at this step. This works well and speeds up the process if the tilt angle used for data collection is pre-defined. The current protocol is written with new specimens in mind, wherein the user may wish to test different tilt angles for data collection.</li>
	<li>Select a Z focus target and regions with holes suitable for high magnification exposures.</li>
	<li>Press Submit targets to queue for imaging. Do not press Submit Queued Targets until finished queuing up all squares.</li>
	<li>Bring the specimen stage back to its untilted state. Move to the next square and repeat steps 2.3-2.8 until an adequate number of hole exposures have been queued.</li>
	<li>Go to the Hole Targeting Node and press Submit Queued Targets once all squares are queued. 
	NOTE: If fine Z focus was disabled previously to save time (step 2.5), it needs to be re-enabled before submitting the queue.</li>
	<li>Manually inspect targets selected by the high magnification Exposure Acquisition node to test if the automated EM hole finder can accurately identify suitable regions for image acquisition when the specimen stage is tilted.
	<ol>
		<li>During this procedure, select &#39;Allow for user verification of selected targets&#39; in Exposure Acquisition node settings. Once the user is satisfied with targeting accuracy, deselect this option for automated data collection. 
		&#8203;NOTE: Targets in high magnification Exposure Acquisition node are typically imaged using a beam-tilt image shift strategy, which works equally well for both tilted and untilted data collection23,24,25,26. For accurate CTF estimation in downstream data processing steps, the lens-coma aberration calibration must be performed for the beam-tilt image shift data collection strategy.</li>
	</ol>
	</li>
</ol>
3. Data Processing
<ol>
	<li>Initiate on-the-fly data processing10,27,28,29 with motion-correction of recordedmovies, CTF estimation, particle selection, and generation of initial reconstructions during data collection. 
	NOTE: For the present study, cryoSPARC Live10 (see Table of Materials) has been utilized for pre-processing. On-the-fly data processing provides an initial cryo-EM reconstruction and an approximation for the angular distribution, which can inform the user about the extent of resolution anisotropy. These can, in turn, be used to guide the user as to whether or not the tilt angle used for data collection is sufficiently high.</li>
	<li>Visualize the reconstructed map and plot the Euler angle distribution to gauge the extent of preferred particle orientations. 
	NOTE: The Euler angle distributions can be converted directly into Fourier space sampling distributions to determine the potential extent of resolution anisotropy. A graphical user interface (GUI) has been developed to assist the user in evaluating the quality of an Euler angle distribution and determining an optimal tilt angle30,31. The tool can be obtained from the Github repository, https://github.com/LyumkisLab/SamplingGui.</li>
	<li>If necessary, adjust the stage tilt angle at which data is collected to overcome the effects of preferential orientation. The angle can be increased if the preferential orientation remains a problem, as evidenced by the map and Euler distribution in 3.2. Alternatively, the user may wish to split the data collection into groups and record using several different tilt angles, such as 20&#176;, 30&#176;, and 40&#176;. 
	NOTE: Although most TEMs must have the capability of tilting the stage to 70&#176;, common specimen stage tilts (that we have used) range from 20&#176;-40&#176;.</li>
</ol>

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The authors have nothing to disclose.

Preferred particle orientation caused by specimen adherence to the air-water interface is one of the last major bottlenecks to routine high-resolution structure determination using cryo-EM SPA4,5,6. The data collection scheme presented here provides an easy-to-implement strategy for improving the orientation distribution of particles within a dataset. We note that the protocol requires no additional equipment or software and doe...

The advent of direct electron detectors over the past decade1,2,3 has spurred an exponential increase in the number of high-resolution structures of macromolecules and macromolecular assemblies solved using single-particle cryo-EM4,5,6. Almost all purified macromolecular species are expected to be amenable to structure determination using cryo-EM, except for the smallest proteins ~10 kDa in size or below7. The amount of starting material needed for grid preparation and structure determination is at least an order of magnitude less than other structure determination techniques, such as nuclear magnetic resonance spectroscopy and X-ray crystallography4,5,6.
However, a principal challenge for structure determination by cryo-EM involves suitable grid preparation for imaging. An extensive study evaluating diverse samples using different vitrification strategies and grids suggested that most approaches for vitrifying samples on cryo-EM grids lead to preferential adherence of macromolecules to the air-water interface8. Such adherence can potentially cause four suboptimal outcomes: (1) the macromolecular sample completely denatures, in which case no successful data collection and processing is possible; (2) the sample partially denatures, in which case it may be possible to obtain structural insights from regions of the macromolecule that are not damaged; (3) the sample retains native structure, but only one set of particle orientations relative to the direction of the electron beam are represented in the images; (4) the sample retains native structure, and some but not all possible particle orientations relative to the direction of the electron beam are represented in the images. For cases (3) and (4), tilted data collection will help with minimizing directional resolution anisotropy affecting the reconstructed cryo-EM map and provides a generalizable solution for a wide variety of samples9. Technically, tilting can also benefit case (2), since the denaturation presumably occurs at the air-water interface and similarly limits the number of distinct orientations represented within the data. The extent of orientation bias in the dataset can potentially be altered by experimenting with solution additives, but a lack of broad applicability hampers these trial-and-error approaches. Tilting the specimen stage at a single optimized tilt angle is sufficient to improve the distribution of orientations by virtue of altering the geometry of the imaging experiment9 (Figure 1). Due to the geometric configuration of the preferentially-oriented sample with respect to the electron beam, for each cluster of preferential orientations, tilting the grid generates a cone of illumination angles with respect to the cluster centroid. Hence, this spreads out the views and consequently improves Fourier space sampling and the isotropy of directional resolution.
There are, in practice, some detriments to tilting the stage. Tilting the specimen stage introduces a focus gradient across the field of view, which may affect the accuracy of contrast transfer function (CTF) estimations. Tilted data collection may also lead to increased beam-induced particle movement caused by increased charging effects when imaging tilted specimens. Grid tilting also leads to an increase in apparent ice thickness, which in turn leads to noisier micrographs and may ultimately impact the resolution of reconstructions5,9,10. It may be possible to overcome these issues by applying advanced computational data-processing schemes that are briefly described in the protocol and discussion sections. Lastly, tilting can lead to increased particle overlap, hindering the subsequent image processing pipeline. Although this can be mitigated to some extent by optimizing on-grid particle concentration, it is nonetheless an important consideration. Here, a simple-to-implement protocol is described for tilted data collection using the Leginon software suite (an automated image acquisition software), available open access and compatible with a broad range of microscopes11,12,13,14. The method requires at least version 3.0 or higher, with versions 3.3 onward containing dedicated improvements to enable tilted data collection. No additional software or equipment is necessary for this protocol. Extensive instructions on computational infrastructure and installation guides are provided elsewhere15.

<table><tbody><tr><td>Cryosparc Live v3.1.0+210216</td><td>Structura Biotechnology</td><td></td><td></td></tr><tr><td>DPS protein</td><td></td><td></td><td>Purification adapted from protocol described in K.Naydenova et al IUCrJ. 2019 Nov 1; 6(Pt 6): 1086&amp;ndash;1098.</td></tr><tr><td>K2 Summit Direct Electron Detector</td><td>Gatan</td><td></td><td></td></tr><tr><td>Leginon software suite</td><td></td><td></td><td>C Suloway et al Journal of Structural Biology 151 (1): pp. 41-60.</td></tr><tr><td>Manual plunging device</td><td></td><td></td><td>Homemade guillotine-like device for vitrification of EM grids</td></tr><tr><td>Talos Arctica</td><td>FEI/Thermo Fisher</td><td></td><td></td></tr><tr><td>UltrAufoil R1.2/1.3 300 mesh grids</td><td>Quantifoil</td><td>N1-A14nAu30-01</td><td></td></tr></tbody></table>

single-particle cryo-em data collection with stage tilt using leginon

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure determination by SPA relies upon obtaining multiple distinct views of a macromolecular object vitrified within a thin layer of ice. Ideally, a collection of uniformly distributed random projection orientations would amount to all possible views of the object, giving rise to reconstructions characterized by isotropic directional resolution. However, in reality, many samples suffer from preferentially oriented particles adhering to the air-water interface. This leads to non-uniform angular orientation distributions in the dataset and inhomogeneous Fourier-space sampling in the reconstruction, translating into maps characterized by anisotropic resolution. Tilting the specimen stage provides a generalizable solution to overcoming resolution anisotropy by virtue of improving the uniformity of orientation distributions, and thus the isotropy of Fourier space sampling. The present protocol describes a tilted-stage automated data collection strategy using Leginon, a software for automated image acquisition. The procedure is simple to implement, does not require any additional equipment or software, and is compatible with most standard transmission electron microscopes (TEMs) used for imaging biological macromolecules.

DPS at 0.3 mg/mL was used to demonstrate imaging at 0&#176;, 30&#176;, and 60&#176; tilts. Data from different tilt angles were collected on the same grid at different grid regions. CTF resolution fits for higher angle tilts tend to be poorer, as was the case when comparing the three datasets in this study. Figure 4 demonstrates comparative representative images and 2D classification averages. Although the protein concentration is unchanged across the different tilt angles, a higher tilt ang...

Watch this Scientific Journal Video about Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon at JoVE.com

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon

The present protocol describes a generalized and easy-to-implement scheme for tilted single-particle data collection in cryo-EM experiments. Such a procedure is especially useful for obtaining a high-quality EM map for samples suffering from preferential orientation bias due to adherence to the air-water interface.

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure ...

single-particle-cryo-em-data-collection-with-stage-tilt-using-leginon

Research

JoVE Journal

Biochemistry

2.3K Views.  The Salk Institute for Biological Studies. The present protocol describes a generalized and easy-to-implement scheme for tilted single-particle data collection in cryo-EM experiments. Such a procedure is especially useful for obtaining a high-quality EM map for samples suffering from preferential orientation bias due to adherence to the air-water interface.

Video: Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon

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, ...

Bioengineering

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.

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 ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

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 ...

Accelerating Macromolecular Structural Analysis Using Smart Leginon Autoscreen

Cryo-Electron Microscopy Screening Automation Across Multiple Grids Using Smart Leginon

Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular protein complexes to near-atomic resolution. The general workflow of the entire cryoEM pipeline involves iterating between sample preparation, cryoEM grid preparation, and sample/grid screening before moving on to high-resolution data collection. Iterating between sample/grid preparation and screening is typically a major bottleneck for researchers, as every iterative experiment must optimize for sample concentration, buffer conditions, grid material, grid hole size, ice thickness, and protein particle behavior in the ice, amongst other variables. Furthermore, once these variables are satisfactorily determined, grids prepared under identical conditions vary widely in whether they are ready for data collection, so additional screening sessions prior to selecting optimal grids for high-resolution data collection are recommended. This sample/grid preparation and screening process often consumes several dozen grids and days of operator time at the microscope. Furthermore, the screening process is limited to operator/microscope availability and microscope accessibility. Here, we demonstrate how to use Leginon and Smart Leginon Autoscreen to automate the majority of cryoEM grid screening. Autoscreen combines machine learning, computer vision algorithms, and microscope-handling algorithms to remove the need for constant manual operator input. Autoscreen can autonomously load and image grids with multi-scale imaging using an automated specimen-exchange cassette system, resulting in unattended grid screening for an entire cassette. As a result, operator time for screening 12 grids may be reduced to ~10 min with Autoscreen compared to ~6 h using previous methods which are hampered by their inability to account for high variability between grids. This protocol first introduces basic Leginon setup and functionality, then demonstrates Autoscreen functionality step-by-step from the creation of a template session to the end of a 12-grid automated screening session.

Author Spotlight: Enhancing Cryo-Electron Microscopy by Automated Data Collection and Analysis Techniques

Cryo-electron microscopy (cryoEM) multi-grid screening is often a tedious process that demands hours of attention. This protocol shows how to set up a standard Leginon collection and Smart Leginon Autoscreen to automate this process. This protocol can be applied to the majority of cryoEM holey foil grids.

Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular ...

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 ...

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 ...

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 ...