This work was supported by the iNEXT-Discovery (Grant 871037) funded by Horizon 2020 program of the European Commission. J B. R. White is funded by the Wellcome Trust (215064/Z/18/Z). The FEI Titan Krios microscopes were funded by the University of Leeds (UoL ABSL award) and Wellcome Trust (108466/Z/15/Z). We thank M Iadanza for use of his micrograph analysis script. We acknowledge Diamond Light Source for access and support of the cryo-EM facilities at the UK&#39;s national Electron Bio-imaging Centre (eBIC) funded by the Wellcome Trust, MRC and BBRSC.

No conflicts of interest are reported.

In this protocol we have described a basic pipeline applicable to specimens amenable to routine SPA. While this filter paper blotting method of thin film formation and vitrification is undoubtedly successful given its use in the vast majority of SPA projects to date, it comes with a number of disadvantages. These include sample wastage, the slow timescales (seconds) required to form the thin film and freeze the specimen, reported irreproducibility27 and reported negative effects of using filter paper to blot away excess liquid50. Recently, new technologies have been developed to improve reproducibility of thin film production51, 52. Other technologies have been developed which reduce the time between sample application and vitrification53,54,55. While filter paper-based methods for thin film formation remain most ubiquitous method of SPA cryoEM sample preparation at the time of writing, these new technologies may bring a range of benefits in terms of efficiency and reproducibility of grid vitrification, as well as creating new opportunities to bring in additional experimental dimensions, such as time resolution and rapid mixing prior to vitrification.
The process of grid screening for most users is presently a qualitative process which involves the acquisition of low magnification atlases followed by taking high-magnification images across the grid to assess particle distribution. While this is a sufficiently robust approach for some types of specimen, it can be difficult to assess by eye if the specimen is indeed what the researcher is hoping to image or has a preferred orientation, for example with small (&#60;200 kDa) samples or where the low-resolution morphology makes it hard to identify by eye if a range of particle distributions are present. For some projects, it is impossible to determine if the specimen is as desired, for example where a ligand is bound or where the sample is being screened to assess if a small (e.g., 10 kDa) subunit is still present in association with a complex. For these projects, fully automated pipelines for data analysis combined with a &#39;short&#39; 0.5 - 1-h collections, that can proceed through image processing steps to 2D classification or even 3D classification and refinement would help efficiently determine if a longer collection is warranted. These pipelines are still under development and are not widely implemented at present, but they have the potential to improve the efficiency of cryoEM grid screening, especially for challenging specimens.
Improvements in direct electron detectors, as well as modifications in microscopy combined with advances in image processing such as image shift data collection, have increased the throughput and quality of images produced during data acquisition. This increase in the rate of data being collected highlights the need for thorough screening of cryoEM grids ahead of many TB of data being acquired.
CryoEM SPA has become a truly mainstream structural biology technique, and in many cases the &#39;go to&#39; approach for some classes of specimens, such as heterogeneous and labile macromolecular complexes. While the protocol here describes a basic overview of the SPA pipeline, each section covered here (grid vitrification and screening, cryoEM and image processing) is a topic in its own right and worthy of exploration during the development of an SPA project. As sample preparation and microscopy technologies progress, and new image processing algorithms and approaches come online, SPA will continue to develop as a pipeline, assisting researchers in gaining insight into complex biological systems.

Single particle CryoEM 
To investigate life at a molecular level we must understand structure. Many techniques to probe protein structure are available, such as NMR, X-ray crystallography, mass spectrometry and electron microscopy (EM). To date, the majority of structures deposited to the Protein Databank (PDB) have been solved using X-ray crystallography. However, from ~2012 onwards, cryo-electron microscopy (cryoEM) became a mainstream technique for protein structure determination and its use increased dramatically. The total number of EM maps deposited to the Electron Microscopy Databank (EMDB) (as of Dec 2020) was 13,421 compared with 1,566 in 2012 (Figure 1, www.ebi.co.uk). In 2012 the number of atomic coordinates modelled in cryoEM density maps, deposited to the PDB was just 67 but as of Dec 2020, 2,309 structures have been deposited so far, a 35-fold increase. This underlying growth in the quality and quantity of cryoEM density maps produced, sometimes referred to as the &#8216;resolution revolution&#39;1, was caused by a coalescence of advances in multiple areas: the development of new cameras for imaging known as direct electron detectors; new software; and more stable&#160;microscopes2,3,4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62415/62415fig01.jpg" /> 
Figure 1: Cumulative submissions to the EMDB from 2012 to December 2020. <a href="https://www.jove.com/files/ftp_upload/62415/62415fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Single particle analysis (SPA) is a powerful tool to generate biological insight in a wide variety of sample types by elucidating high resolution structures of isolated complexes5, 6 including viruses7,8, membrane proteins9, 10, helical assemblies11 and other dynamic and heterogeneous macromolecular complexes12, 13, the sizes of which vary by orders of magnitude (from 39 kDa14, 15 to tens of megadaltons). Here, a protocol for a standard pipeline for cryoEM SPA from sample to structure is described.
Prior to embarking upon this pipeline, a purified sample should be subjected to biochemical analysis to assess its chances of downstream success. Preparation of a suitable sample is arguably the biggest barrier to SPA, particularly for transient and heterogeneous (both compositional and conformational) complexes. The macromolecular complex preparation should contain as few contaminants as possible, at sufficient concentration to yield many particles in each cryoEM micrograph, and in a buffer composition well suited to cryoEM analysis. Certain buffer constituents, including sucrose, glycerol and high (~&#62; 350 mM concentrations of salts, depending on the sample size, properties and other buffer constituents) can interfere with the process of vitrification or reduce the signal-to-noise ratio in images, hindering structure determination16.
Typically, as a minimum, size exclusion chromatography (SEC) and SDS- PAGE gel analysis should be used to assess sample purity17, 18, but circular dichroism, functional assays, SEC coupled with multi-angle light scattering, and thermal stability assays are all useful tools for qualitative analysis of macromolecular complex preparations prior to cryoEM analysis. However, the results from these biochemical analyses may yield little insight into the structural heterogeneity of the sample and its behaviour on a cryoEM grid. For this reason, negative stain EM is routinely used as a quick, cheap and powerful tool for assessing compositional and conformational heterogeneity, and therefore a good way of ascertaining which elution fraction from a purification is most promising, or screening different buffer compositions19, 20. Once a promising sample has been identified, we can proceed to the SPA cryoEM pipeline. Negative stain does not always align with the subsequent results seen in cryoEM; sometimes a sample looks poor by negative stain but improves when seen in vitreous ice in cryoEM. In contrast, sometimes samples look excellent during negative stain steps but require significant further optimisation when progressing to cryoEM. However, in the majority of cases negative stain provides a useful quality control step.
Vitrification 
The harsh environment within the vacuum system of the electron microscope causes both dehydration and radiation damage to unfixed biological specimens21. Therefore, to image the sample in a native-like state, the biological specimen must be preserved prior to imaging. For purified preparations of macromolecular complexes, vitrification is the method of choice to enable its visualization by cryoEM whilst preserving the atomic details of the complex. The discovery of vitrification as a method of sample preparation was a fundamental advancement in electron microscopy of biological specimens, for which Dubochet was recognized in the 2017 Nobel Prize in Chemistry. Sample vitrification involves creating a thin layer of solution containing the specimen of interest, typically tens of nm thick, suspended on a cryoEM grid support. The thin film is then frozen extremely rapidly in a cryogen such as liquid ethane at ~-175 &#176;C. The freezing rate is ~106 &#176;C/s, fast enough that amorphous, or vitreous ice forms, suspending the specimen in a thin, solid film22.
The initial variable to consider is the cryoEM grid support chosen23. An EM grid typically consists of an amorphous carbon film with perforations (either regular or irregular), over a support structure. The support structure is typically a circular metal grid 3.05 mm in diameter, usually made from copper, but other metals such as gold, or molybdenum (which has preferred thermal expansion properties24) can be used. Sometimes, an additional thin, continuous support is applied across the grid, such as graphene, graphene oxide or a thin (~1-2 nm) amorphous carbon layer. While standard cryoEM grids (most commonly 400-200 mesh copper with a perforated (1.2 &#181;m round holes separated by 1.3 &#181;m (r1.2/1.3), or 2 &#181;m separated by 2 &#181;m of carbon (r2/2)) carbon support- although many different patterns are available) have been used in the vast majority of structures reported to date, novel grid technologies with improved conductivity and reduced specimen movement have been reported25. Selected grids are subjected to a glow-discharge/plasma cleaning treatment to render them hydrophilic and amenable to sample application26.
Following glow-discharge, the next stage is thin film formation. This thin film is most commonly formed using filter paper to remove excess liquid from the grid. While this can be carried out manually, a number of plunge freezing devices are commercially available, including the Vitrobot Mk IV (Thermo Fisher Scientific), EM GP II (Leica) and CP3 (Gatan). With these devices, ~3-5 &#181;L of sample in solution is applied to the EM grid, followed by blotting away excess solution using filter paper. The grid, with a thin film suspended across it, is then plunged into liquid ethane cooled by liquid nitrogen (LN2) to ~-175 &#176;C. Once frozen, the grid is maintained at a temperature below the devitrification point (-137 &#176;C) prior to, and during imaging.
Specimen screening and data collection 
Following vitrification of a cryoEM grid, the next stage is to screen the grid to assess its quality and to determine whether the grid is suitable to proceed to high resolution data collection. An ideal cryoEM grid has vitreous ice (opposed to crystalline ice) with the ice thickness just sufficient to accommodate the longest dimension of the specimen, ensuring the surrounding ice contributes as little noise to the resulting image as possible. The particles within the ice should have a size and (if known) shape consistent with biochemistry, and ideally be monodisperse with a random distribution of particle orientations. Finally, the grid should have enough areas of sufficient quality to satisfy the desired data collection length. Depending on the specimen, this may take many iterations of vitrification and screening until optimal grids are produced. Both fortunately and unfortunately, there are a huge range of variables that can be empirically tested to alter particle distribution on cryoEM grids (reviewed in16,27). In this manuscript, representative results for a membrane protein project10 are shown.
Once a suitable grid has been identified, data collection can proceed. Several models of cryo-transmission electron microscopes for biological specimens are optimized to collect high-resolution data in an automated fashion. Typically, data is collected on 300 kV or 200 kV systems. Automated data collection can be achieved using software including EPU (Thermo Fisher Scientific)28, Leginon29, JADAS30&#160;and SerialEM31, 32. An automated data collection with modern detectors typically results in terabytes (TB) of raw data in a 24 h period (average datasets are ~ 4 TB in size).
Due to the COVID-19 restrictions in place on much of the world (time of writing December 2020), many microscopy facilities have moved to offering remote access. Once the grids have been loaded into the autoloader of a microscope, data acquisition can be conducted remotely.
Image processing and model building 
Where a data collection session may be typically 0.5-4 days, subsequent image processing may take many weeks and months, depending on the availability of computing resources. It is standard for initial image processing steps, namely motion correction and contrast transfer function (CTF) estimation to take place &#39;on the fly&#39; 33, 34. For downstream processing, there are a plethora of software suites available. Particles are &#39;picked&#39; and extracted from micrographs35, 36. Once particles are extracted, a standard protocol would be to process the particles through several rounds of classification (in both two dimensions (2D) and three dimensions (3D) and/or focused on specific regions of interest) to reach a homogeneous subset of particles. This homogeneous subset of particles is then averaged together to produce a 3D reconstruction. At this point data is often corrected further to produce the highest quality map possible, for example through CTF refinement, distortion corrections37&#160;and Bayesian polishing38&#160;. The outcome of this image processing is a 3D cryoEM map of the biological specimen of interest. The resolution range reached in a &#39;standard&#39; automated single particle experiment from a grid of sufficient quality, with data collected on a 300 kV microscope system is typically between 10 &#197; and 2 &#197; depending on the size and flexibility of the protein complex. With an ideal specimen, resolutions of ~1.2 &#197; have now been reached using SPA workflows5. While this protocol details steps towards obtaining an EM density map, once this is in hand it can be further interpreted through fitting and refining a protein model (if resolution is &#60; 3.5 &#197;) or building de novo39. Data associated with structure determination experiments can be deposited into online public repositories, including EM density maps (Electron Microscopy Data Bank)40, resulting atomic coordinates (Protein Data Bank)41&#160;and raw datasets (Electron Microscopy Public Image Archive)42.
In this protocol, the outer-membrane protein complex RagAB (~340 kDa) from Porphyromonas gingivalis is used as an example macromolecular complex10 (EMPIAR-10543). For those new to cryoEM, support for samples through this pipeline from sample to structure is available, subject to peer review, through funded access schemes such as iNEXT Discovery and Instruct.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blunt tweezers</td>
			<td>Agar Scientific</td>
			<td>AGT5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo EM round storage box</td>
			<td>Agar Scientific</td>
			<td>AGG3736</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoEM autogrid boxes</td>
			<td>ThermoFisher Scientific</td>
			<td>1084591</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoEM grids</td>
			<td>Quantifoil</td>
			<td>N1-C14nCu30-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethane gas</td>
			<td>Boc</td>
			<td>270595-F</td>
			<td></td>
		</tr>
		<tr>
			<td>LN2&#160; foam dewar</td>
			<td>Agar Scientific</td>
			<td>AG81760-500</td>
			<td></td>
		</tr>
		<tr>
			<td>LN2&#160; storage dewar</td>
			<td>Worthington industries</td>
			<td>HC 34</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Gilson</td>
			<td>10082012</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Star labs</td>
			<td>s1111-1706</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>BD 300869</td>
			<td></td>
		</tr>
		<tr>
			<td>Type II lab water</td>
			<td>Suez</td>
			<td>select fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitrobot</td>
			<td>ThermoFisher Scientific</td>
			<td>1086439</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitrobot filter paper</td>
			<td>Whatman</td>
			<td>1001-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitrobot styrophome container assembly</td>
			<td>ThermoFisher Scientific</td>
			<td>1086439</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitrobot tweesers</td>
			<td>ThermoFisher Scientific</td>
			<td>72882-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EPU</td>
			<td>ThermoFisher Scientific</td>
			<td>2.8.1.10REL</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM server</td>
			<td>ThermoFisher Scientific</td>
			<td>6.15.3.22415REL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tia</td>
			<td>ThermoFisher Scientific</td>
			<td>5.0.0.2896REL</td>
			<td></td>
		</tr>
		<tr>
			<td>Titan krios microscope</td>
			<td>ThermoFisher Scientific</td>
			<td>Titan Krios G2</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

When screening, grids can be discarded at the atlas stage, where features resolved at low magnification mark the grid as not suitable for data acquisition. For example, if a grid has been subject to significant mechanical damage with the majority of grid squares broken (Figure 2A), or where the grid appears to be &#39;dry&#39;, with no vitreous ice (Figure 2B). Such grids are typically identifiable as the edges of the grid squares appear sharp and distinct. Across the majority of grids made using the plunge freezing device, a gradient of ice is observed (Figure 2C,D). Particle distribution, depending on the specimen of interest, can vary dramatically with ice thickness and so screening a range of grid squares to assess particle distribution is recommended. Tools have been implemented within EPU software during the atlas screening step to help the user identify grid squares of similar or different ice thickness, which can be particularly useful to users who are new to examining cryoEM grids (Figure 2E, F).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62415/62415fig02.jpg" /> 
Figure 2: Example low magnification &#39;atlas&#39; montages from screening sessions. A) A grid which has suffered significant damage with the majority of grid squares broken - unsuitable for collection. B) A dry grid with no vitreous ice - unsuitable for collection. C) A grid demonstrating an ice gradient with ~ 50% of the grid useable. D) An ice gradient with ~ 33% of the grid useable. Both C and D, are suitable for data collection if the usable grid squares have an ice thickness appropriate for collection, and there are enough acquisition areas to satisfy the minimum duration of a collection (e.g., 24 h) E) An example atlas with range of ice thicknesses. F) The same atlas presented in E but with, grid squares categorized and coloured by EPU software according to ice thickness. <a href="https://www.jove.com/files/ftp_upload/62415/62415fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
When screening particle distribution, ensure that imaging parameters, such as magnification and total electron dose, are similar to those expected to be used during data acquisition in order to provide an accurate picture of expected results. During screening, an ideal particle distribution is monodisperse with a range of particle orientations visible (depending on the specimen and existing knowledge of the particle&#39;s morphology, this may be challenging to ascertain) (Figure 3A). The ice should be as thin as possible while accommodating the particles largest dimension, if ice is too thin it can melt when illuminated with the electron beam. This causes excessive motion in the micrograph, and areas that display this characteristic should be avoided (Figure 3B). From collective experience, this effect is most commonly observed when there is detergent in the buffer. This can result in very thin ice at the centre&#160;of the hole and so particles can be physically excluded and forced towards the edge. This effect is observed in Figure 3C, but in this case it is not an extreme example and these images would still usefully contribute to a dataset. Finally, the ice needs to be vitreous; exclude any areas of the grid (or grids) where the majority or all of the images taken show crystalline ice (Figure 3D) from data acquisition. Often, non-vitreous ice is observed at the edge of grid squares. Readers are referred to detailed reviews of the variables that can be altered during grid vitrification16 and descriptions of particle behaviour in the thin film environment46, 47&#160;for further information.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62415/62415fig03.jpg" /> 
Figure 3: Representative micrographs showing differing particle distributions. A) An &#39;ideal&#39; distribution of monodisperse particles adopting a range of orientations. B) Overly thin ice in the middle of the hole that it deforms upon exposure to the electron beam causing excessive motion in the micrograph. This effect is most often observed when detergent is present in the buffer C) Where ice is thinner in the centre of the hole, this physically excludes particles from the centre, causing crowding of particles towards the hole edge. In this case it is not extreme enough to prevent these images being useful, but it suggests it is worth screening slightly thicker areas. D) Ice is not vitreous, data should not be collected on areas which look like this example micrograph. <a href="https://www.jove.com/files/ftp_upload/62415/62415fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
On-the-fly image processing can help to pick up errors and problems with data acquisition and so is always recommended where possible. For example, excessive motion within micrographs may indicate that the autoloader turbo pump is active, or data is being collected on a cracked grid square where ice is moving significantly in the electron beam, indicating the grid square should be skipped. On the fly CTF estimation can reveal circumstances where a positive focus point (rather than defocus) is applied (where CTF estimation programs and parameters to find these points are used), and determine the phase shift where a Volta phase plate48&#160;is used. On the fly image processing pipelines often include a graphical summary of the data (Figure 4A) to make it easier for users to assess micrograph quality quickly and decide if data collection amendments are required.
Selection of particles from micrographs, whilst avoiding &#39;false positives&#39; such as contamination or the grid support film can require optimisation. However, particle pickers such as crYOLO often work sufficiently well using default parameters for a &#39;first pass&#39; of the data (Figure 4B), enabling progression to 2D class averaging where it can be easier to assess the quality of the data and the likelihood of downstream success. For most projects, 2D classification of ~&#62; 10k particles should start to reveal classes which have secondary structure detail. To proceed to 3D, the 2D classification stage should typically reveal classes representing a range of particle orientations. If a preferred orientation is revealed, more iterations of sample preparation16 or further data acquisition with the sample tilted may be required49. All classes which show secondary structure detail should be chosen to take forward to 3D analysis, while &#39;junk&#39; particles are discarded (Figure 4C).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62415/62415fig04.jpg" /> 
Figure 4: Initial image processing steps. A) Output from an &#39;on the fly&#39; image processing script. B) Example micrograph (left) with appropriately auto-picked particles identified using the crYOLO general model (right, with particles bounded by red squares) Scale bars (white) are 50 nm. C) Results from 2D classification showing classes which were discarded in the red square, and classes from which particles were selected for further processing in green. <a href="https://www.jove.com/files/ftp_upload/62415/62415fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A small subset of particles can be used to generate an initial model (Figure 5A). This initial model can then be used as a starting model in 3D classification and refinement. In the case of RagAB, the dataset contained three distinct conformers which can be separated during 3D classification (Figure 5B). Particles contributing to each of these classes can then be treated independently and used to refine an EM density map which can then be subject to further interpretation and model building.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62415/62415fig05.jpg" /> 
Figure 5: Generating 3D EM density map. A) Typical initial model generated using RELION. B) 3D classification over 5 classes showing separation of particles into three distinct conformational states: open-open (green), open-closed (blue), closed-closed (purple). C) Process of mask creation. The map from 3D refinement (left) should be visualized in chimera. The volume viewer can then be used to identify the lowest threshold at which the map is free from disjointed, noisy density (middle). This threshold value is input as initial binarization threshold in the RELION Mask creation job. An example mask output is shown in grey (right). D) High resolution EM density map of the open-closed state of RagAB (EMD-10245), filtered and colored by local resolution (&#197;).&#160;<a href="https://www.jove.com/files/ftp_upload/62415/62415fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Single Particle Cryo-Electron Microscopy: From Sample to Structure at JoVE.com

Single Particle Cryo-Electron Microscopy: From Sample to Structure

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 ...

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8.2K Views.  University of Leeds. 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.

Video: Single Particle Cryo-Electron Microscopy: From Sample to Structure

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 ...

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 ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

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 ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

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 ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

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 ...

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase &#946; and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

This protocol outlines in detail the preparation of nucleosomal complexes using two methods of sample preparation for freezing TEM grids.

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of ...