Better, Faster, Cheaper: Recent Advances in Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.

A novel bluetongue virus serotype 2 strain isolated from a farmed Florida white-tailed deer (Odocoileus virginianus) arose from reassortment of gene segments derived from co-circulating serotypes in the Southeastern USA.

Bluetongue disease is a reportable animal disease that affects wild and farmed ruminants, including white-tailed deer (WTD). This report documents the clinical findings, ancillary diagnostics, and genomic characterization of a novel reassortant bluetongue virus serotype 2 (BTV-2) strain isolated from a dead Florida farmed WTD in 2022. Our analyses support that this BTV-2 strain likely stemmed from the acquisition of genome segments from co-circulating BTV strains in Florida and Louisiana. In addition, our analyses also indicate that genetically uncharacterized BTV strains may be circulating in the Southeastern USA; however, the identity and reassortant status of these BTV strains cannot be determined based on the VP2 and VP5 genome sequences. Hence, continued surveillance based on complete genome characterization is needed to understand the genetic diversity of BTV strains in this region and the potential threat they may pose to the health of deer and other ruminants.

Smart data collection for CryoEM.

This report provides an overview of the discussions, presentations, and consensus thinking from the Workshop on Smart Data Collection for CryoEM held at the New York Structural Biology Center on April 6-7, 2022. The goal of the workshop was to address next generation data collection strategies that integrate machine learning and real-time processing into the workflow to reduce or eliminate the need for operator intervention.

Variations in Gut Microbiota of Siberian Flying Squirrels Correspond to Seasonal Phenological Changes in Their Hokkaido Subarctic Forest Ecosystem.

Gut microbial communities of animals are influenced by diet and seasonal weather changes. Since foraging strategies of wild animals are affected by phenological changes, gut microbial communities would differ among seasons. However, interactions of plant-animal-microbiota with seasonal changes have not been well characterized. Here, we surveyed gut microbial diversity of Siberian flying squirrels (Pteromys volans orii) from a natural forest in Hokkaido during spring and summer of 2013 and 2014. Additionally, we compared microbial diversity to temperature changes and normalized difference vegetation index (NDVI). Changes in both seasonal temperature and phenology were significantly associated with alterations in gut microbiota. There were two clusters of OTUs, below and above 20 °C that were significantly correlated with low and high temperatures, respectively. Low-temperature cluster OTUs belonged to various phyla, whereas the high-temperature cluster was only constituted by Firmicutes. In conclusion, gut microbiota of Siberian flying squirrels varied with environmental changes on an ecological scale.

Routine determination of ice thickness for cryo-EM grids.

Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.

High resolution single particle cryo-electron microscopy using beam-image shift.

Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (±0.1 µm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam-image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.

Best practices for managing large CryoEM facilities.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

MRC2014: Extensions to the MRC format header for electron cryo-microscopy and tomography.

The MRC binary file format is widely used in the three-dimensional electron microscopy field for storing image and volume data. Files contain a header which describes the kind of data held, together with other important metadata. In response to advances in electron microscopy techniques, a number of variants to the file format have emerged which contain useful additional data, but which limit interoperability between different software packages. Following extensive discussions, the authors, who represent leading software packages in the field, propose a set of extensions to the MRC format standard designed to accommodate these variants, while restoring interoperability. The MRC format is equivalent to the map format used in the CCP4 suite for macromolecular crystallography, and the proposal also maintains interoperability with crystallography software. This Technical Note describes the proposed extensions, and serves as a reference for the standard.

CTF Challenge: Result summary.

Image formation in bright field electron microscopy can be described with the help of the contrast transfer function (CTF). In this work the authors describe the "CTF Estimation Challenge", called by the Madrid Instruct Image Processing Center (I2PC) in collaboration with the National Center for Macromolecular Imaging (NCMI) at Houston. Correcting for the effects of the CTF requires accurate knowledge of the CTF parameters, but these have often been difficult to determine. In this challenge, researchers have had the opportunity to test their ability in estimating some of the key parameters of the electron microscope CTF on a large micrograph data set produced by well-known laboratories on a wide set of experimental conditions. This work presents the first analysis of the results of the CTF Estimation Challenge, including an assessment of the performance of the different software packages under different conditions, so as to identify those areas of research where further developments would be desirable in order to achieve high-resolution structural information.

Multiplexed TEM Specimen Preparation and Analysis of Plasmonic Nanoparticles.

We describe a system for rapidly screening hundreds of nanoparticle samples using transmission electron microscopy (TEM). The system uses a liquid handling robot to place up to 96 individual samples onto a single standard TEM grid at separate locations. The grid is then transferred into the TEM and automated software is used to acquire multiscale images of each sample. The images are then analyzed to extract metrics on the size, shape, and morphology of the nanoparticles. The system has been used to characterize plasmonically active nanomaterials.

Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200 kV.

A new era has begun for single particle cryo-electron microscopy (cryoEM) which can now compete with X-ray crystallography for determination of protein structures. The development of direct detectors constitutes a revolution that has led to a wave of near-atomic resolution cryoEM reconstructions. However, regardless of the sample studied, virtually all high-resolution reconstructions reported to date have been achieved using high-end microscopes. We demonstrate that the new generation of direct detectors coupled to a widely used mid-range electron microscope also enables obtaining cryoEM maps of sufficient quality for de novo modeling of protein structures of different sizes and symmetries. We provide an outline of the strategy used to achieve a 3.7 Å resolution reconstruction of Nudaurelia capensis ω virus and a 4.2 Å resolution reconstruction of the Thermoplasma acidophilum T20S proteasome.

Recent advances in data collection for Cryo-EM methods.

Methods of transmission electron microscopy (TEM) are typically used to resolve structures of vitrified biological specimens using both single particle analysis (SPA) and tomographic methods and use both conventional as well as scanning transmission modes of data collection. Automation of data collection for each method has been developed to different levels of convenience for the users. Automation of methods using the conventional TEM mode has progressed the furthest. Beam-image shift strategies first used in data collection for SPA were shown to be equally valuable for cryo-electron tomography (cryo-ET). Machine learning methods have been applied for target selection and for planning optimal paths of data collection for SPA. These methods also enabled automated screening. Apertures matching the square shape of cameras have been recently described. Some progress has also been made in the automation of cryo applications of scanning TEM, promising an increase of throughput and potential for further improvement.

Mycobacterium kansasii Infection in a Farmed White-Tailed Deer (Odocoileus virginianus) in Florida, USA.

A 7-year-old farmed white-tailed deer doe was transported to a Levy County, Florida property and began to decline in health, exhibiting weight loss and pelvic limb weakness. The doe prematurely delivered live twin fawns, both of which later died. The doe was treated with corticosteroids, antibiotics, gastric cytoprotectants, and B vitamins but showed no improvement. The doe was euthanized, and a post mortem examination was performed under the University of Florida's Cervidae Health Research Initiative. We collected lung tissue after the animal was euthanized and performed histological evaluation, using H&E and Ziehl-Neelsen (ZN) staining, and molecular evaluation, using conventional PCR, followed by Sanger sequencing. The microscopic observations of the H&E-stained lung showed multifocal granuloma, while the ZN-stained tissue revealed low numbers of beaded, magenta-staining rod bacteria inside the granuloma formation. Molecular analysis identified the presence of Mycobacterium kansasii. This isolation of a non-tuberculous Mycobacterium in a white-tailed deer emphasizes the importance of specific pathogen identification in cases of tuberculosis-like disease in farmed and free-ranging cervids. We report the first case of M. kansasii infection in a farmed white-tailed deer (Odocoileus virginianus) in Florida. Although M. kansasii cases are sporadic in white-tailed deer, it is important to maintain farm biosecurity and prevent farmed cervids from contacting wildlife to prevent disease transmission.

Maximizing the potential of electron cryomicroscopy data collected using direct detectors.

Single-particle electron cryomicroscopy is undergoing a technical revolution due to the recent developments of direct detectors. These new recording devices detect electrons directly (i.e. without conversion into light) and feature significantly improved detective quantum efficiencies and readout rates as compared to photographic films or CCDs. We evaluated here the potential of one such detector (Gatan K2 Summit) to enable the achievement of near-atomic resolution reconstructions of biological specimens when coupled to a widely used, mid-range transmission electron microscope (FEI TF20 Twin). Compensating for beam-induced motion and stage drift provided a 4.4Å resolution map of Sulfolobus turreted icosahedral virus (STIV), which we used as a test particle in this study. Several motion correction and dose fractionation procedures were explored and we describe their influence on the resolution of the final reconstruction. We also compared the quality of this data to that collected with a FEI Titan Krios microscope equipped with a Falcon I direct detector, which provides a benchmark for data collected using a high-end electron microscope.

An integrated micro- and macroarchitectural analysis of the Drosophila brain by computer-assisted serial section electron microscopy.

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.

Learning to automate cryo-electron microscopy data collection with Ptolemy.

Over the past decade, cryo-electron microscopy (cryoEM) has emerged as an important method for determining near-native, near-atomic resolution 3D structures of biological macromolecules. To meet the increasing demand for cryoEM, automated methods that improve throughput and efficiency of microscope operation are needed. Currently, the targeting algorithms provided by most data-collection software require time-consuming manual tuning of parameters for each grid, and, in some cases, operators must select targets completely manually. However, the development of fully automated targeting algorithms is non-trivial, because images often have low signal-to-noise ratios and optimal targeting strategies depend on a range of experimental parameters and macromolecule behaviors that vary between projects and collection sessions. To address this, Ptolemy provides a pipeline to automate low- and medium-magnification targeting using a suite of purpose-built computer vision and machine-learning algorithms, including mixture models, convolutional neural networks and U-Nets. Learned models in this pipeline are trained on a large set of images from real-world cryoEM data-collection sessions, labeled with locations selected by human operators. These models accurately detect and classify regions of interest in low- and medium-magnification images, and generalize to unseen sessions, as well as to images collected on different microscopes at another facility. This open-source, modular pipeline can be integrated with existing microscope control software to enable automation of cryoEM data collection and can serve as a foundation for future cryoEM automation software.

Fully automated multi-grid cryoEM screening using Smart Leginon.

Single-particle cryo-electron microscopy (cryoEM) is a swiftly growing method for understanding protein structure. With increasing demand for high-throughput, high-resolution cryoEM services comes greater demand for rapid and automated cryoEM grid and sample screening. During screening, optimal grids and sample conditions are identified for subsequent high-resolution data collection. Screening is a major bottleneck for new cryoEM projects because grids must be optimized for several factors, including grid type, grid hole size, sample concentration, buffer conditions, ice thickness and particle behavior. Even for mature projects, multiple grids are commonly screened to select a subset for high-resolution data collection. Here, machine learning and novel purpose-built image-processing and microscope-handling algorithms are incorporated into the automated data-collection software Leginon, to provide an open-source solution for fully automated high-throughput grid screening. This new version, broadly called Smart Leginon, emulates the actions of an operator in identifying areas on the grid to explore as potentially useful for data collection. Smart Leginon Autoscreen sequentially loads and examines grids from an automated specimen-exchange system to provide completely unattended grid screening across a set of grids. Comparisons between a multi-grid autoscreen session and conventional manual screening by 5 expert microscope operators are presented. On average, Autoscreen reduces operator time from ∼6 h to <10 min and provides a percentage of suitable images for evaluation comparable to the best operator. The ability of Smart Leginon to target holes that are particularly difficult to identify is analyzed. Finally, the utility of Smart Leginon is illustrated with three real-world multi-grid user screening/collection sessions, demonstrating the efficiency and flexibility of the software package. The fully automated functionality of Smart Leginon significantly reduces the burden on operator screening time, improves the throughput of screening and recovers idle microscope time, thereby improving availability of cryoEM services.

Beam-induced motion of vitrified specimen on holey carbon film.

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 µm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.

Precise beam-tilt alignment and collimation are required to minimize the phase error associated with coma in high-resolution cryo-EM.

Electron microscopy at a resolution of 0.4nm or better requires more careful adjustment of the illumination than is the case at a resolution of 0.8nm. The use of current-axis alignment is not always sufficient, for example, to avoid the introduction of large phase errors, at higher resolution, due to axial coma. In addition, one must also ensure that off-axis coma does not corrupt the data quality at the higher resolution. We particularly emphasize that the standard CTF correction does not account for the phase error associated with coma. We explain the cause of both axial coma and the typically most troublesome component of off-axis coma in terms of the well-known shift of the electron diffraction pattern relative to the optical axis that occurs when the illumination is not parallel to the axis. We review the experimental conditions under which coma causes unacceptably large phase errors, and we discuss steps that can be taken when setting up the conditions of illumination, so as to ensure that neither axial nor off-axis coma is a problem.

Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy.

We report on initial results of using a new direct detection device (DDD) for single particle reconstruction of vitreous ice embedded specimens. Images were acquired on a Tecnai F20 at 200keV and a nominal magnification of 29,000×. This camera has a significantly improved signal to noise ratio and modulation transfer function (MTF) at 200keV compared to a standard CCD camera installed on the same microscope. Control of the DDD has been integrated into Leginon, an automated data collection system. Using GroEL as a test specimen, we obtained images of ∼30K particles with the CCD and the DDD from the same specimen sample using essentially identical imaging conditions. Comparison of the maps reconstructed from the CCD images and the DDD images demonstrates the improved performance of the DDD. We also obtained a 3D reconstruction from ∼70K GroEL particles acquired using the DDD; the quality of the density map demonstrates the potential of this new recording device for cryoEM data acquisition.