We thank D. Roderer for providing us TcdA1 micrographs. We thank Steve Ludtke for his ongoing support of EMAN2 infrastructure. This work was supported by funds from the Max Planck Society (to S.R.) and the European Council under the European Union's Seventh Framework Programme (FP7/2007-2013) (grant no. 615984) (to S.R.) and grant from the National Institutes of Health R01 GM60635 to P.A.P.).

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The authors declare that they have no competing financial interests.

Single particle cryo-EM has shown a rapid development in the recent years and delivered numerous atomic resolution structures of macromolecular complexes of major biological significance25. In order to support the large number of novice users that are currently entering the field, we developed the single particle image analysis platform SPHIRE and present here a walk-through protocol for the entire workflow including movie alignment, particle picking, CTF estimation, initial model calculation, 2D and 3D heterogeneity analysis, high-resolution 3D refinement and local resolution estimation and filtering.
The protocol described here is intended as a short guide to 3D structure determination using cryo-EM micrographs of the protein of interest and with the help of computational tools provided by the stand-alone GUI of SPHIRE.
The main feature of the workflow is that most of the procedures need to be run only once, since they rely on the concept of validation by reproducibility19 and do not require parameter tweaking. This automatic validation mechanism is a main advantage of SPHIRE over other software packages since the results tend to be objective as well as reproducible and, most importantly, obtainable at an acceptable computational cost. The pipeline provides in addition a wealth of diagnostic information for experienced users to conduct further independent validation and assessment with own methods. Nevertheless, a novice user who has at least elemental theoretical background in structural biology and electron microscopy should be able to obtain near-atomic resolution structures using own data and the automated validation procedures.
However, obtaining a near-atomic resolution structure is not always straightforward and the result will highly depend on the quality of the sample and the input data. For the procedures presented here, it is assumed that a sufficient number of high quality unaligned raw EM movies are available, with their averages showing clearly discernible homogeneous and randomly orientated single particles. In general, there are no restrictions regarding symmetry, size or overall shape of the molecule, but a low molecular weight can be a limiting factor, especially when the protein has a featureless globular shape. Usually, analysis of larger, well-ordered particles with high point-group symmetry is less demanding. Therefore, it is strongly recommend for novice users to run the present protocol first with a well-characterized cryo-EM dataset. Either the SPHIRE tutorial data (http:/sphire.mpg.de) or one of the EMPIAR submitted datasets (https://www.ebi.ac.uk/pdbe/emdb/empiar/) with raw movies are a good starting point.
When processing own data, it is very likely that some datasets or some of the images will not satisfy certain quality criteria. In this context, in addition to the automated stability and reproducibility checks, performed by the program for major steps of the workflow, it is still recommend for users to visually inspect the results at certain &#34;checkpoints&#34; of the protocol, especially if the final reconstruction is not satisfactory.
The first visual inspection can be done at the micrograph level after the movie alignment (Protocol step 2) and the CTF estimation (Protocol step 3). The resulting motion-corrected averages should show clearly discernible and well-separated single particles and their power spectra should show clearly discernible, isotropic Thon rings. The spatial frequency to which they are visible defines, in most cases, the highest resolution to which the structure can in principle be ultimately determined. Examples of a motion-corrected average of sufficient quality and its power spectrum are shown in the section &#34;Representative results&#34;. Outlier images that might have a negative impact on the final result can be removed with the help of SPHIRE&#39;s Drift and CTF assessment GUI tools (http://sphire.mpg.de/wiki/doku.php).
With regard to particle screening, the crucial step in the SPHIRE pipeline is the 2D classification using ISAC (Protocol step 5.2). Here, the user should control that the reproducible 2D class averages identified automatically by the program adopt a range of orientations sufficient to quasi-evenly cover the angular space. If the quality of the class averages is not satisfactory (noisy and/or blurry images) and/or the number of reproducible class averages is very low, consider improving the auto-picking quality, optimizing dataset imaging or sample preparation. In most cases, it is not possible to calculate a reliable reconstruction from a dataset that does not yield good 2D class averages. Examples of high quality 2D class averages are shown in the section &#34;Representative results&#34;.
At least 100 class averages are required to obtain a reliable initial 3D model using RVIPER in an automated manner (Protocol step 6.1). For this step, the user should select the averages with the highest quality and include as many different orientations of the particle as possible. The quality of the initial model is critical for the success of the subsequent high-resolution 3D refinement.
In other software packages, 3D classification is sometimes performed to remove &#34;bad&#34; particles8,9. However, in SPHIRE most of these particles are automatically eliminated already during 2D classification using ISAC. Thus, it is recommended to perform the computationally intensive step of 3D sorting only if the reconstruction and the 3D variability analysis indicate heterogeneity of the dataset.
Most importantly, the user should always carefully inspect the resulting 3D volumes carefully (Protocol step 9.3), and confirm that the features of the respective density agree well with the nominal resolution. At a resolution of &#60;9 &#197;, rod-like densities corresponding to &#945;-helices become visible. At a resolution &#60;4.5 &#197;, densities corresponding to strands in &#946;-sheets are normally well separated and bulky amino acids become visible. A high-resolution map (&#60;3 &#197;) should show clearly discernible side chains, thus allowing building of an accurate atomic model.
Results obtained to date demonstrate that, with the help of SPHIRE&#39;s automated reproducibility tests and minimal visual inspections, the present protocol is generally applicable to any type of single particle cryo-EM project. Representative results of each processing step are shown for the reconstruction of the TcdA1 toxin of Photorhabdus luminescens 21, which has been solved to near-atomic resolution. Density maps of similar quality can be used to construct reliable atomic models by de novo backbone tracing as well as reciprocal or real-space refinement, and thus provide a solid structural framework for the understanding of complex molecular mechanisms.
ACCESION CODES:
The coordinates for the EM structure and the unprocessed movies have been deposited in the Electron Microscopy Data Bank and the Electron Microscopy Pilot Image Archive under accession numbers EMD-3645 and EMPIAR-10089, respectively.

After the development of the direct electron detector technology, the remarkable progress in single particle cryo-EM is currently reshaping structural biology 1. Compared with X-ray crystallography, this technique requires only a small amount of protein material without the need for crystallization, while simultaneously posing fewer restrictions regarding purity of the sample and still allowing determination of structures at near-atomic resolution. Importantly, different compositions or states can now be computationally separated and structure determination of the different conformations can be carried out at unprecedented level of detail. Recently, density maps of challenging molecules could be produced at resolutions allowing de novo model building and thus deep understanding of their mode of action 2,3,4,5.
A wide variety of image processing software packages are available in the 3DEM (3D Electron Microscopy) community (https://en.wikibooks.org/wiki/Software_Tools_For_Molecular_Microscopy) and most of them are under continuous development. Near-atomic resolution has been achieved for proteins exhibiting various molecular weights and symmetries with several different software packages, including EMAN2 6, IMAGIC7, FREALIGN 8, RELION 9, SPIDER 10, and SPARX 11. Each package requires a different level of user expertise and provides a different level of user guidance, automation and extensibility. Moreover, whereas some programs provide complete environments to facilitate all steps of image analysis, others are designed to optimize specific tasks, such as the refinement of alignment parameters starting from a known reference structure. More recently, several platforms have been developed, including APPION 12 and SCIPION 13, that provide a single processing pipeline which integrates approaches and protocols from the different software packages listed above.
To contribute to the current development of cryo-EM, SPARX was re-developed into a new stand-alone and complete platform for single particle analysis, called SPHIRE (SPARX for High-Resolution Electron Microscopy). In order to increase accessibility of the technique for new researchers in the field and to cope with the large amount of data produced by modern fully-automated high-end electron microscopes, the processing pipeline was redesigned and simplified by introducing an easy-to-use Graphical User Interface (GUI) and automating the major steps of the workflow. Moreover, new algorithms were added to allow fast, reproducible and automated structure determination from cryo-EM images. Furthermore, validation by reproducibility was introduced in order to avoid common artifacts produced during refinement and heterogeneity analysis.
Although the program was extensively modified, its appreciated core features were maintained: straightforward open-source code, the modern object-oriented design and Python interfaces for all basic functions. Thus, it was not changed into a black box program, enabling users to study and easily modify the Python code, to create additional applications or modify the overall workflow. This is especially useful for non-standard cryo-EM projects.
Here we present a protocol for obtaining a near-atomic resolution density map from cryo-EM images using the GUI of SPHIRE. It describes in detail all steps required to generate a density map from raw cryo-EM direct detector movies and is not restricted to any particular macromolecule type. This protocol primarily intends to guide newcomers in the field through the workflow and provide important information about crucial steps of the processing as well as some of the possible pitfalls and obstacles. More advanced features and the theoretical background behind SPHIRE will be described elsewhere.

<table><tbody><tr><td>SPHIRE</td><td>Max Planck Institute of Molecular Physiology- Dortmund&amp;nbsp; and Houston Medical School, Houston, Texas&amp;nbsp;</td><td></td><td>http://sphire.mpg.de</td></tr><tr><td>UCSF Chimera</td><td>University of California, San Francisco</td><td></td><td>http://www.cgl.ucsf.edu/chimera/</td></tr><tr><td>Unblur</td><td>Janelia Farm Research Campus, Ashburn</td><td></td><td>http://grigoriefflab.janelia.org/unblur</td></tr><tr><td>Coot</td><td>MRC Laboratory of Molecular Biology,&amp;nbsp; Cambridge</td><td></td><td>http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/</td></tr><tr><td>EMAN2</td><td>Baylor College of Medicine, Houston</td><td></td><td>http://blake.bcm.edu/emanwiki/EMAN2</td></tr><tr><td>Computing Cluster with 1824 cores</td><td>Max Planck Institute of Molecular Physiology</td><td></td><td>Linux Cluster with 76&amp;nbsp; nodes, each with 2 Processors Xeon E5-2670v3 12C 2.30 GHz and 128 Gb RAM</td></tr><tr><td>TITAN KRIOS electron microscope&amp;nbsp;</td><td>FEI</td><td></td><td>300 kV, Cs correction, XFEG</td></tr><tr><td>Falcon II direct electron detector</td><td>FEI</td><td></td><td></td></tr><tr><td>EPU (automated data acquisition software)</td><td>FEI</td><td></td><td>https://www.fei.com/software/epu/</td></tr></tbody></table>

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.

The protocol described above was executed starting from 112 direct detector movies of the A component of the Photorhabdus luminescens Tc complex (TcdA1) 20,21,22. This dataset was recorded on a Cs-corrected electron cryo-microscope with a high-brightness field emission gun (XFEG), operated at an acceleration voltage of 300 kV. The images were acquired automatically with a total dose of 60 e-/&#197;-2&#160;at a pixel size of 1.14 &#197; on the specimen scale. After alignment of the movie frames (Protocol Step 2), the resulting motion-corrected averages had isotropic Thon rings extending to high-resolution (Figure 2a). The individual particles were easily visible and well separated (Figure 2b). Particles were then picked using the swarm tool of e2boxer 18 (Protocol Step 4.1). In this case, an appropriate threshold was set using the more selective option (Figure 2c). The 112 digital micrographs yielded 9,652 particles. The majority of the extracted images (Protocol Step 4.2) contained well-defined particles and their box size was ~1.5 times larger than the particle size, as recommended (Figure 2d). Next, using ISAC, a 2D heterogeneity analysis was performed (Protocol Step 5). It yielded 98 class averages (Figure 3a). Using these 2D class averages, an ab initio model was calculated using VIPER (Protocol Step 6) at intermediate resolution (Figure 3b). This model shows excellent agreement with the crystal structure of TcdA1 previously solved at 3.9 &#197; resolution 22 (Figure 3c). This ab initio model was used as an initial template for the 3D refinement (MERIDIEN), yielding a 3.5 &#197; (0.143 criterion) reconstruction (Protocol Step 7) from only ~40,000 asymmetric units (Figure 4). This near-atomic resolution map was obtained within 24 h, using up to 96 CPUs for the steps of the workflow that benefit from multiple cores.
For the 3D variability analysis (Protocol step 8), only 2,000 particle images per group were used in step 8.3.3 (i.e. the process starts with 5 initial 3D groups) and 200 images for the smallest group size in step 8.3.4 due to the small number of particles (~10,000). The analysis revealed localized flexibility mainly at the N-terminal region of the complex that contains the His tag used for purification (Figure 5a). Indeed, twelve N-terminal residues and the His tag were not resolved in the previously published crystal structure of TcdA1 22 and this most probably disordered region remained unresolved in the present cryo-EM density, likely due to its flexibility. Additional variability was detected at the receptor-binding domains and the BC-binding domain (Figure 5a). Due to the overall satisfactory resolution of the structure and the rather small size of the dataset, this heterogeneity was decided to be tolerable and therefore a focused 3D classification 23 was not performed. Finally, the local resolution of the final density map was computed (Protocol step 9.1, Figure 5b) and the sharpened 3D map was locally filtered (Protocol step 9.2). A volume of this quality can be used for de novo model building using Coot 24 or any other refinement tool (Figure 6).
<img alt="Figure 1" src="/files/ftp_upload/55448/55448fig1.jpg" /> 
Figure 1: Image Processing using SPHIRE. (a) The GUI of the SPHIRE software package. A specific step of the workflow can be activated by selecting the respective pictogram on the left side of the GUI (&#34;workflow step&#34;). The commands and utilities associated with this step of the workflow will appear in the central area of the GUI. After selecting one of the commands, the respective parameters are shown on the right area of the GUI. Advanced parameters usually do not require modification of the preset default values. (b) Stages in the workflow of single particle image processing using the SPHIRE GUI. <a href="http://cloudflare.jove.com/files/ftp_upload/55448/55448fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55448/55448fig2.jpg" /> 
Figure 2: Motion Correction and Particle Extraction. (a, b) Typical high-quality, low-dose, drift-corrected digital micrograph recorded at a defocus of 1.7 &#181;m. Note the isotropic Thon rings extending to a resolution of 2.7 &#197; in the power spectrum (a) and the well discernible particles in the 2D image (b). (c) Particle selection using e2boxer. Green circles indicate selected particles. (d) Typical raw particles extracted from the dose-weighted micrograph. Scale bars = 20 nm. <a href="http://cloudflare.jove.com/files/ftp_upload/55448/55448fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55448/55448fig3.jpg" /> 
Figure 3: 2D Clustering and Initial Model Generation. (a) Gallery of 2D class averages, with the majority representing side views of the particle. Scale bar = 20 nm. (b) Ab initio 3D map of TcdA1 obtained using RVIPER from the reference-free class averages. (c) Rigid-body fitting of the TcdA1 crystal structure (ribbons) (pdb-id 1VW1) into the initial cryo-EM density (transparent gray). <a href="http://cloudflare.jove.com/files/ftp_upload/55448/55448fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55448/55448fig4.jpg" /> 
Figure 4: Cryo-EM 3D Structure of TcdA1. (a, b) Final 3.5 &#197; density map of TcdA1 computed using ~9,500 particle images: (a) side and (b) top view. (c) Representative areas of the cryo-EM density for an &#945;-helix and a &#946;-sheet. <a href="http://cloudflare.jove.com/files/ftp_upload/55448/55448fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55448/55448fig5.jpg" /> 
Figure 5: Variability Analysis and Local Resolution. (a) Surface of the sharpened TcdA1 cryo-EM map (gray) and the variability map (green). For better clarity, the variability map was low-pass filtered to 30 &#197;. (b) Surface rendering of the TcdA1 sharpened cryo-EM map colored according to local resolution (&#197;). Note the topological agreement between areas of high variability and low local resolution. <a href="http://cloudflare.jove.com/files/ftp_upload/55448/55448fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55448/55448fig6.jpg" /> 
Figure 6: 3D Model Building of TcdA1 using Coot. Representative regions of the cryo-EM density and the atomic model are shown for an &#945;-helix. The atomic model was built de novo using Coot. <a href="http://cloudflare.jove.com/files/ftp_upload/55448/55448fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

high-resolution-single-particle-analysis-from-electron-cryo

Nanyang Technological University

Research

JoVE Journal

Biochemistry

50.2K Views.  Max Planck Institute of Molecular Physiology. 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.

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

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

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 ...

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.

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 ...

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 ...

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 ...

Coating CryoEM Grids with Monolayer Graphene to Improve Cryo-Sample Preparation

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 &#181;m wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM&#39;s widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 &#197; resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

Author Spotlight: Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids 

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...

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 ...

Immunology and Infection

Enhancing Cryo-EM Map Quality with CryoSieve Particle Selection

Enhancing Density Maps by Removing the Majority of Particles in Single Particle Cryogenic Electron Microscopy Final Stacks

Over the past decade, advancements in technology and methodology within the field of cryogenic electron microscopy (cryo-EM) single-particle analysis (SPA) have substantially improved our capacity for high-resolution structural examination of biological macromolecules. This advancement has ushered in a new era of molecular insights, replacing X-ray crystallography as the dominant method and providing answers to longstanding questions in biology. Since cryo-EM does not depend on crystallization, which is a significant limitation of X-ray crystallography, it captures particles of varying quality. Consequently, the selection of particles is crucial, as the quality of the selected particles directly influences the resolution of the reconstructed density map. An innovative iterative approach for particle selection, termed CryoSieve, significantly improves the quality of reconstructed density maps by effectively reducing the number of particles in the final stack. Experimental evidence shows that this method can eliminate the majority of particles in final stacks, resulting in a notable enhancement in the quality of density maps. This article outlines the detailed workflow of this approach and showcases its application on a real-world dataset.

Author Spotlight: Optimizing Cryo-EM Analysis with CryoSieve for Enhanced Particle Selection Efficiency

An advanced particle selection method for cryo-EM, namely CryoSieve, improves density map resolution by removing a majority of particles in final stacks, as demonstrated through its application on a real-world dataset.

Over the past decade, advancements in technology and methodology within the field of cryogenic electron microscopy (cryo-EM) single-particle analysis ...

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

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