The authors would like to acknowledge economical support from: The Spanish Ministry of Science and Innovation through Grants: PID2019-104757RB-I00/AEI/10.13039/501100011033, the &#34;Comunidad Aut&#243;noma de Madrid&#34; through Grant: S2017/BMD-3817, Instituto de Salud Carlos III, PT17/0009/0010 (ISCIII-SGEFI/ERDF), European Union (EU) and Horizon 2020 through grant: INSTRUCT - ULTRA (INFRADEV-03-2016-2017, Proposal: 731005), EOSC Life (INFRAEOSC-04-2018, Proposal: 824087), iNEXT - Discovery (Proposal: 871037), and HighResCells (ERC - 2018 - SyG, Proposal: 810057). The project that gave rise to these results received the support of a fellowship from &#34;la Caixa&#34; Foundation (ID 100010434). The fellowship code is LCF/BQ/DI18/11660021. This project has received funding from the European Union&#39;s Horizon 2020 research and innovation programme under the Marie Sk&#322;odowska-Curie grant agreement No. 713673. The authors acknowledge the support and the use of resources of Instruct, a Landmark ESFRI project.

<ol>
	<li>Nogales, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+cryo-EM+into+a+mainstream+structural+biology+technique.">The development of cryo-EM into a mainstream structural biology technique.</a> Nature Methods. 13 (1), 24-27 (2016).</li><li>K&#252;hlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Resolution+Revolution.">The Resolution Revolution.</a> Science. 343 (6178), 1443-1444 (2014).</li><li>. 1.15 A structure of human apoferritin obtained from Titan Mono- BCOR microscope Available from: <a target="_blank" href="https://www.rcsb.org/structure/7A6A">https://www.rcsb.org/structure/7A6A</a> (2021)</li><li>Arnold, S. 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A., Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+determination+of+local+defocus+and+specimen+tilt+in+electron+microscopy.">Accurate determination of local defocus and specimen tilt in electron microscopy.</a> Journal of Structural Biology. 142, 334-347 (2003).</li><li>Winn, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+CCP4+suite+and+current+developments.">Overview of the CCP4 suite and current developments.</a> Acta crystallographica. 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The authors have nothing to disclose.

Currently, cryo-EM is a key tool to reveal the 3D structure of biological samples. When good data is collected with the microscope, the available processing tools will allow us to obtain a 3D reconstruction of the macromolecule under study. Cryo-EM data processing is able to achieve near-atomic resolution, which is key to understanding the functional behavior of a macromolecule and is also crucial in drug discovery.
Scipion is a software that allows creating the whole workflow combining the most relevant image processing packages in an integrative way, which helps the traceability and reproducibility of the entire image-processing workflow. Scipion provides a very complete set of tools to carry out the processing; however, obtaining high resolutions reconstructions depends completely on the quality of the acquired data and how these data is processed. 
To get a high resolution 3D reconstruction, the first requirement is to obtain good movies from the microscope, which preserve structural information to high resolution. If this is not the case, the workflow will not be able to extract high definition information from the data. Then, a successful processing workflow should be able to extract particles that really correspond to the structure and to find the orientations of these particles in the 3D space. If any of the steps in the workflow fails, the quality of the reconstructed volume will be degraded. Scipion allows for using different packages in any of the processing steps, which helps to find the most adequate approach to process the data. Moreover, thanks to having many packages available, consensus tools, that boost the accuracy by finding an agreement in the estimated outputs of different methods, can be used. Also, it has been discussed in detail in the Representative Results section several validation tools and how to identify accurate and inaccurate results in every step of the workflow, to detect potential problems, and how to try to solve them. There are several checkpoints along the protocol that could help to realize if the protocol is running properly or not. Some of the most relevant are: picking, 2D classification, initial volume estimation, and 3D alignment. Checking the inputs, repeating the step with a different method, or using consensus, are options available in Scipion that the user can use to find solutions when issues appear. 
Regarding the previous approaches to package integration in the Cryo-EM field, Appion31 is the only one that allows real integration of different software packages. However, Appion is tightly connected with Leginon32, a system for automated collection of images from electron microscopes. The main difference with Scipion is that data model and storage are less coupled. In such a way, to create a new protocol in Scipion, only a Python script needs to be developed. However, in Appion, the developer must write the script and change the underlying database. In summary, Scipion was developed to simplify maintenance and extensibility.
We have presented in this manuscript a complete workflow for Cryo-EM processing, using the real case dataset of the Plasmodium falciparum 80S Ribosome (EMPIAR entry: 10028, EMDB entry: 2660). The steps covered and discussed here can be summarized as movie alignment, CTF estimation, particle picking, 2D classification, initial map estimation, 3D classification, 3D refinement, evaluation, and post-processing. Different packages have been used and consensus tools were applied in several of these steps. The final 3D reconstructed volume achieved a resolution of 3 &#197; and, in the post-processed volume, some secondary structures can be distinguished, like alpha-helices, which helps to describe how atoms are arranged in space.
The workflow presented in this manuscript shows how Scipion can be used to combine different Cryo-EM packages in a straightforward and integrative way to simplify the processing, and obtain more reliable result at the same time.
In the future, the development of new methods and packages will keep growing and software like Scipion to easily integrate all of them will be even more important for the researchers. Consensus approaches will be more relevant even then, when plenty of methods with different basis will be available, helping to obtain more accurate estimations of all the parameters involve in the reconstruction process in Cryo-EM. Tracking and reproducibility are key in the research process and easier to achieve with Scipion thanks to having a common framework for the execution of complete workflows.

In cryo-electron microscopy (cryo-EM), single particle analysis (SPA) of vitrified frozen-hydrated specimens is one of the most widely used and successful variants of imaging for biological macromolecules, as it allows to understand molecular interactions and the function of biological ensembles1. This is thanks to the recent advances in this imaging technique that gave rise to the "resolution revolution"2 and have allowed the successful determination of biological 3D structures with near-atomic resolution. Currently, the highest resolution achieved in SPA cryo-EM was 1.15 &#197; for apoferritin3 (EMDB entry: 11668). These technological advances comprise improvements in the sample preparation4, the image acquisition5, and the image processing methods6. This article is focused on this last point. 
Briefly, the goal of the image processing methods is to identify all the acquisition parameters to invert the imaging process of the microscope and recover the 3D structure of the biological specimen under study. These parameters are the gain of the camera, the beam-induced movement, the aberrations of the microscope (mainly the defocus), the 3D angular orientation and translation of each particle, and the conformational state in case of having a specimen with conformational changes. However, the number of parameters is very high and cryo-EM requires using low-dose images to avoid radiation damage, which significantly reduces the Signal-to-Noise Ratio (SNR) of the acquired images. Thus, the problem cannot be unequivocally solved and all the parameters to be calculated only can be estimations. Along the image processing workflow, the correct parameters should be identified, discarding the remaining ones to finally obtain a high-resolution 3D reconstruction.
The data generated by the microscope are gathered in frames. Simplifying, a frame contains the number of electrons that have arrived at a particular position (pixel) in the image, whenever electron-counting detectors are used. In a particular field of view, several frames are collected and this is called a movie. As low electron doses are used to avoid radiation damage that could destroy the sample, the SNR is very low and the frames corresponding to the same movie need to be averaged to obtain an image revealing structural information about the sample. However, not only a simple average is applied, the sample can suffer shifts and other kinds of movements during the imaging time due to the beam-induced movement that need to be compensated. The shift-compensated and averaged frames originate a micrograph.
Once the micrographs are obtained, we need to estimate the aberrations introduced by the microscope for each of them, called Contrast Transfer Function (CTF), which represents the changes in the contrast of the micrograph as a function of frequency. Then, the particles can be selected and extracted, which is called particle picking. Every particle should be a small image containing only one copy of the specimen under study. There are three families of algorithms for particle picking: 1) the ones that only use some basic parameterization of the appearance of the particle to find them in the whole set of micrographs (e.g., particle size), 2) the ones that learn how the particles look like from the user or a pretrained set, and 3) the ones that use image templates. Each family has different properties that will be shown later.
The extracted set of particles found in the micrographs will be used in a 2D classification process that has two goals: 1) cleaning the set of particles by discarding the subset containing pure noise images, overlapping particles, or other artifacts, and 2) the averaged particles representing each class could be used as initial information to calculate a 3D initial volume. 
The 3D initial volume calculation is the next crucial step. The problem of obtaining the 3D structure can be seen as an optimization problem in a multidimensional solution landscape, where the global minimum is the best 3D volume that represents the original structure, but several local minima representing suboptimal solutions can be found, and where it is very easy to get trapped. The initial volume represents the starting point for the searching process, so bad initial volume estimation could prevent us to find the global minimum. From the initial volume, a 3D classification step will help to discover different conformational states and to clean again the set of particles; the goal is to obtain a structurally homogeneous population of particles. After that, a 3D refinement step will be in charge of refining the angular and translation parameters for every particle to get the best 3D volume possible. 
Finally, in the last steps, the obtained 3D reconstruction can be sharpened and polished. Sharpening is a process of boosting the high frequencies of the reconstructed volume, and the polishing is a step to further refine some parameters, as CTF or beam-induced movement compensation, at the level of particles. Also, some validation procedures could be used to better understand the achieved resolution at the end of the workflow. 
After all these steps, the tracing and docking processes7 will help to give a biological meaning to the obtained 3D reconstruction, by building atomic models de novo or fitting existing models. If high resolution is achieved, these processes will tell us the positions of the biological structures, even of the different atoms, in our structure.
Scipion8 allows creating the whole workflow combining the most relevant image processing packages in an integrative way. Xmipp9, Relion10, CryoSPARC11, Eman12, Spider13, Cryolo14, Ctffind15, CCP416, Phenix17, and many more packages can be included in Scipion. Also, it incorporates all the necessary tools to benefit the integration, interoperability, traceability, and reproducibility to make a full tracking of the entire image-processing workflow8.
One of the most powerful tools that Scipion allows us to use is the consensus, which means to compare the results obtained with several methods in one step of the processing, making a combination of the information conveyed by different methods to generate a more accurate output. This could help to boost the performance and improve the achieved quality in the estimated parameters. Note that a simpler workflow can be build without the use of consensus methods; however, we have seen the power of this tool22,25 and the workflow presented in this manuscript will use it in several steps. 
All the steps that have been summarized in the previous paragraphs will be explained in detail in the following section and combined in a complete workflow using Scipion. Also, how to use the consensus tools to achieve a higher agreement in the generated outputs will be shown. To that end, the example dataset of the Plasmodium falciparum 80S Ribosome has been chosen (EMPIAR entry: 10028, EMDB entry: 2660). The dataset is formed by 600 movies of 16 frames of size 4096x4096 pixels at a pixel size of 1.34&#197; taken at an FEI POLARA 300 with an FEI FALCON II camera, with a reported resolution at EMDB is 3.2&#197;18 .

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>no material is used in this article</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

cryo-em and single-particle analysis with scipion

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.

Watch this Scientific Journal Video about Cryo-EM and Single-Particle Analysis with Scipion at JoVE.com

Cryo-EM and Single-Particle Analysis with Scipion

Single-particle analysis in cryo-electron microscopy is one of the main techniques used to determine the structure of biological ensembles at high resolution. Scipion provides the tools to create the whole pipeline to process the information acquired by the microscope and achieve a 3D reconstruction of the biological specimen.

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at ...

cryo-em-and-single-particle-analysis-with-scipion

Research

JoVE Journal

Biochemistry

3.6K Views.  Campus Universidad Autónoma de Madrid. Single-particle analysis in cryo-electron microscopy is one of the main techniques used to determine the structure of biological ensembles at high resolution. Scipion provides the tools to create the whole pipeline to process the information acquired by the microscope and achieve a 3D reconstruction of the biological specimen.

Video: Cryo-EM and Single-Particle Analysis with Scipion

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

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 ...

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method for structural biologists to determine high-resolution structures of a wide variety of macromolecules. Multiple software suites are available to new and expert users for image processing and structure calculation, which streamline the same basic workflow: movies acquired by the microscope detectors undergo correction for beam-induced motion and contrast transfer function (CTF) estimation. Next, particle images are selected and extracted from averaged movie frames for iterative 2D and 3D classification, followed by 3D reconstruction, refinement, and validation. Because various software packages employ different algorithms and require varying levels of expertise to operate, the 3D maps they generate often differ in quality and resolution. Thus, users regularly transfer data between a variety of programs for optimal results. This paper provides a guide for users to navigate a workflow across the popular software packages: cryoSPARC v3, RELION-3, and Scipion 3 to obtain a near-atomic resolution structure of the adeno-associated virus (AAV). We first detail an image processing pipeline with cryoSPARC v3, as its efficient algorithms and easy-to-use GUI allow users to quickly arrive at a 3D map. In the next step, we use PyEM and in-house scripts to convert and transfer particle coordinates from the best quality 3D reconstruction obtained in cryoSPARC v3 to RELION-3 and Scipion 3 and recalculate 3D maps. Finally, we outline steps for further refinement and validation of the resultant structures by integrating algorithms from RELION-3 and Scipion 3. In this article, we describe how to effectively utilize three processing platforms to create a single and robust workflow applicable to a variety of data sets for high-resolution structure determination.

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

This article describes how to effectively utilize three cryo-EM processing platforms, i.e., cryoSPARC v3, RELION-3, and Scipion 3, to create a single and robust workflow applicable to a variety of single-particle data sets for high-resolution structure determination.

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...

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 ...

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers of multi-layered samples. This removal of layers prior to plunge freezing can aid in reducing sample thickness to a level suitable for cryo-EM data collection, improving sample flatness, and facilitating image processing. The peel-blot technique allows for the separation of multilamellar membranes into single layers, of layered 2D crystals into individual crystals, and of stacked, sheet-like structures of soluble proteins to likewise be separated into single layers. The high sample thickness of these types of samples frequently poses insurmountable problems for cryo-EM data collection and cryo-EM image processing, especially when the microscope stage must be tilted for data collection. Furthermore, grids of high concentrations of any of these samples can be prepared for efficient data collection since sample concentration prior to grid preparation can be increased and the peel-blot technique adjusted to result in a dense distribution of single-layered specimen.

The peel-blot technique is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...

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.

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 ...