Abstract Images in the electron microscope are formed by electron scattering and focusing. The spherical geometry of these processes gives rise to two coherent, conjugate spherical wave fronts, known as Ewald spheres. These spheres are associated with the two halves of the contrast transfer function (CTF), and their widths are determined by the focal gradient through the specimen. To properly correct for the CTF, each half of the CTF must be applied to an image individually and integrated into the reconstruction into the corresponding Ewald sphere. Theory indicates that this dual Ewald sphere reconstruction method should recover the maximal amount of information possible. This method was compared to the other reconstruction methods commonly used: the projection approximation (ignoring the Ewald sphere), the simple insertion and the single sideband methods. In simulated reconstructions the dual Ewald sphere method recovered the most information when the correct half of the CTF is matched to the corresponding Ewald sphere. If the wrong half is matched, the result worse than the projection approximation method. Examining reconstructions from real data indicated that the dual Ewald sphere method performs at least as well as the simple insertion method, but not as good as in simulations. The likely reason is the two-fold ambiguity in the assigned orientations of the particle images, which remains an issue to pursue in further studies. In conclusion, the dual Ewald sphere reconstruction method may offer the best way to calculate very high resolution reconstructions when the micrograph quality warrants it. Highlights The dual Ewald sphere reconstruction corrects for the two halves of the CTF. The signs of the two halves of the CTF must correspond to the focal gradient. Determining the focal gradient for individual particle images remains unresolved. Complex reconstructions indicate any real space phases are artifacts.

Herpesviruses are prevalent infectious agents in humans, with complex structures and life cycles. The viability and detail of a model of capsid assembly and maturation can now be examined against the recently available mature herpesvirus capsids structures. The first large assembly product is the icosahedral procapsid with an outer shell composed of major capsid proteins (MCPs) connected by triplexes (heterotrimers composed of one Tri1 protein and two Tri2 proteins), and an inner shell of scaffold proteins. The asymmetric triplexes have specific and conserved orientations, suggesting a key role in assembly. In the mature capsid structures, triplexes bound to three MCPs may represent an assembly unit where, in most cases, the N-terminus of one MCP wraps around the E-loop of another MCP. The model accommodates the incorporation of a portal into capsid, required for genome encapsidation and viral viability. Cleavage of the scaffold triggers maturation of procapsid. Each of the MCPs rotates mostly as a rigid body, except for the flexible peripheral parts that remodel to close the capsid inner surface. Angularization of the capsid shifts the portal outward to a better contact with the capsid shell. Understanding these events in the herpesvirus life cycle to atomic detail could facilitate the development of drugs that uniquely target assembly and maturation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Retinoschisin (RS1) is a secreted protein that is essential for maintaining integrity of the retina. Numerous mutations in RS1 cause X-linked retinoschisis (XLRS), a progressive degeneration of the retina that leads to vision loss in young males. A key manifestation of XLRS is the formation of cavities (cysts) in the retina and separation of the layers (schisis), disrupting synaptic transmission. There are currently no approved treatments for patients with XLRS. Strategies using adeno-associated viral (AAV) vectors to deliver functional copies of RS1 as a form of gene augmentation therapy, are under clinical evaluation. To improve therapeutic strategies for treating XLRS, it is critical to better understand the secretion of RS1 and its molecular function. Immunofluorescence and immunoelectron microscopy show that RS1 is located on the surfaces of the photoreceptor inner segments and bipolar cells. Sequence homology indicates a discoidin domain fold, similar to many other proteins with demonstrated adhesion functions. Recent structural studies revealed the tertiary structure of RS1 as two back-to-back octameric rings, each cross-linked by disulfides. The observation of higher order structures in vitro suggests the formation of an adhesive matrix spanning the distance between cells (∼100 nm). Several studies indicated that RS1 readily binds to other proteins such as the sodium-potassium ATPase (NaK-ATPase) and extracellular matrix proteins. Alternatively, RS1 may influence fluid regulation via interaction with membrane proteins such as the NaK-ATPase, largely inferred from the use of carbonic anhydrase inhibitors to shrink the typical intra-retinal cysts in XLRS. We discuss these models in light of RS1 structure and address the difficulty in understanding the function of RS1.

In our quest to solve biomolecular structures to higher resolutions in cryoEM, care must be taken to deal with all aspects of image formation in the electron microscope. One of these is the Ewald sphere/focus gradient that derives from the scattering geometry in the microscope and its implications for recovering high resolution and handedness information. While several methods to deal with it has been proposed and implemented, there are still questions as to the correct approach. At the high acceleration voltages used for cryoEM, the traditional projection approximation that ignores the Ewald sphere breaks down around 2-3 Å and with large particles. This is likely not crucial for most biologically interesting molecules, but is required to understand detail about catalytic events, molecular orbitals, orientation of bound water molecules, etc. Through simulation I show that integration along the Ewald spheres in frequency space during reconstruction, the “simple insertion method” is adequate to reach resolutions to the Nyquist frequency. Both theory and simulations indicate that the handedness information encoded in such phases is irretrievably lost in the formation of real space images. The conclusion is that correct reconstruction along the Ewald spheres avoids the limitations of the projection approximation.

Journal Article Accounting for the Ewald Spheres in CryoEM Reconstructions and Their Relationship to 3D Fourier Transforms of Focal Series Get access J Bernard Heymann, J Bernard Heymann National Cryo-EM Program, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA Search for other works by this author on: Oxford Academic Google Scholar Alan Merk, Alan Merk National Cryo-EM Program, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA Search for other works by this author on: Oxford Academic Google Scholar Jana Ognjenovic Jana Ognjenovic National Cryo-EM Program, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Pages 1030–1031, https://doi.org/10.1093/micmic/ozad067.523 Published: 22 July 2023

From the first isolation of the cystovirus bacteriophage Φ6 from Pseudomonas syringae 50 years ago, we have progressed to a better understanding of the structure and transformations of many parts of the virion. The three-layered virion, encapsulating the tripartite double-stranded RNA (dsRNA) genome, breaches the cell envelope upon infection, generates its own transcripts, and coopts the bacterial machinery to produce its proteins. The generation of a new virion starts with a procapsid with a contracted shape, followed by the packaging of single-stranded RNA segments with concurrent expansion of the capsid, and finally replication to reconstitute the dsRNA genome. The outer two layers are then added, and the fully formed virion released by cell lysis. Most of the procapsid structure, composed of the proteins P1, P2, P4, and P7 is now known, as well as its transformations to the mature, packaged nucleocapsid. The outer two layers are less well-studied. One additional study investigated the binding of the host protein YajQ to the infecting nucleocapsid, where it enhances the transcription of the large RNA segment that codes for the capsid proteins. Finally, I relate the structural aspects of bacteriophage Φ6 to those of other dsRNA viruses, noting the similarities and differences.

Bsoft is a software package primarily developed for processing electron micrographs, with the goal of determining the structures of biologically relevant molecules, molecular assemblies, and parts of cells. However, it incorporates many ways to deal with images, from the mundane to very sophisticated algorithms. This article is an introduction into its use, illustrating that it is an extensive toolbox, for manipulating and understanding images. Bsoft has over 150 programs, allowing the user an infinite number of ways to process images. These programs can be executed on the command line, or through the interactive program called brun. The main visualization program is bshow, providing numerous ways to manipulate and interpret images. The primary aim is to provide the user with powerful capabilities, including processing large numbers of images. An important additional aim is to make it as accessible as possible, making it easier to deal with image formats and features, and enhance productivity.

Background and Objective: The quality of a cryoEM reconstruction is fundamentally a function of the signal-to-noise ratio (SNR) of the original micrographs. The SNR embodies multiple aspects of image formation, including microscope details and alignment, specimen composition and thickness, how it is recorded, and how the specimen degrades during imaging. With the advent of direct electron detectors and the recording of a series of images for each micrograph (a movie), we have an opportunity to count every electron and derive fully quantitative results. After alignment of the movie frames of a micrograph, we can calculate the SNR, or its spatial frequency equivalent, the spectral SNR (SSNR). This SSNR reflects residual movement between frames and the progressive effect of radiation damage. The goal is to develop a quantitative analysis of the SSNR and radiation damage to assess and improve the quality of micrographs.Methods: Several test cases were selected from the EMPIAR database and ten micrograph movies downloaded for each case. The movie frames were aligned as rigid bodies to compensate for stage and support movement. The SSNR for subsets of frames was then calculated to assess the effect of residual movement. The progressive SSNR (PSSNR) was subsequently calculated to determine the decrease in signal accumulation as a result of radiation damage. Results: In all cases the alignment of the movie frames compensated for global movement to the extent that the effect on the SSNR is negligible. The subset SSNR can be used as a tool to further confirm the extent of residual movement. The progressive SSNR indicates an increase in value up to an asymptote, consistent with the theory for radiation damage. Fitting these curves gives the inherent SNR before exposure, and the critical dose, which decreases with spatial frequency with an exponential parameter roughly between one and two.Conclusions: The implementation of the PSSNR for movie frames provides a tool for assessing micrograph quality and progression of radiation damage. The estimation of the critical dose further quantifies radiation damage and may shed some light on the mechanisms of damage. These are likely both a function of the specimen composition and the imaging parameters used.Published by Elsevier B.V.

In recent years, advances in cryoEM have dramatically increased the resolution of reconstructions and, with it, the number of solved atomic models. It is widely accepted that the quality of cryoEM maps varies locally; therefore, the evaluation of the maps-derived structural models must be done locally as well. In this article, a method for the local analysis of the map-to-model fit is presented. The algorithm uses a comparison of two local resolution maps. The first is the local FSC (Fourier shell correlation) between the full map and the model, while the second is calculated between the half maps normally used in typical single particle analysis workflows. We call the quality measure “FSC-Q”, and it is a quantitative estimation of how much of the model is supported by the signal content of the map. Furthermore, we show that FSC-Q may be helpful to detect overfitting. It can be used to complement other methods, such as the Q-score method that estimates the resolvability of atoms.

Herpes simplex virus 1 (HSV-1) infects a majority of humans, causing mostly mild disease but in some cases progressing toward life-threatening encephalitis. Understanding the life cycle of the virus is important to devise countermeasures.

The goal of cryoEM is to determine the structures of biomolecules from electron micrographs. In many cases the processing is straightforward and can be handled with routine protocols. In other cases, the properties and behavior of the specimen require adaptions to properly interpret the data. Here I describe the protocols for examining the higher order assemblies of the retinal adhesion protein, retinoschisin (RS1), using the Bsoft package. The protocols for micrograph preprocessing, 2D classification and 3D alignment and reconstruction follow the usual patterns for the majority of cryoEM specimens. The interpretation of the results is specific to the branched network of RS1 filaments. The 2D class averages are used to determine the relative positions of the RS1 molecules, thus defining the interacting interfaces in the network. The major interface of the linear filament is then further examined by reconstructing the "unit cell" and fitting the molecular models.

“Giant” phages have genomes of >200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them “mottled capsids”. Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a “twisted hook” conformation. Large patches of a proteinaceous tetragonal lattice with a 67 Å repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.

Bsoft offers many tools for the processing of tomographic tilt series and the interpretation of tomograms. Since I introduced tomography into Bsoft almost two decades ago, the field has advanced significantly, requiring refinement of old algorithms and development of new ones. The current direct detectors allow us to collect data more efficiently and with better quality, progressing towards automation. The goal is then to also automate alignment of tilt series and reconstruction. I added an estimation of the specimen thickness as well as fiducialless alignment, to augment the existing fiducial-based alignment. High-resolution work requires correction for the contrast transfer function, in tomography complicated by the tilted specimen. For this, I developed a method to generate a power spectrum using the whole micrograph, compensating for tilting. This is followed by routine determination of the contrast transfer function, and correction for it during reconstruction. The next steps involve interpretation of the tomogram, either by subtomogram averaging where possible, or by segmentation and modeling otherwise. Such interpretation actually constitutes the main time-consuming part of tomography and is less amenable to automation compared to the initial reconstruction.

In single-particle analysis (SPA), the aim is to obtain a 3D reconstruction of a biological molecule from 2D electron micrographs to the highest level of detail or resolution as possible. Current practice is to collect large volumes of data, hoping to reach high-resolution maps through sheer numbers. However, adding more particles from a specific data set eventually leads to diminishing improvements in resolution. Understanding what these resolution limits are and how to deal with them are important in optimization and automation of SPA. This study revisits the theory of 3D reconstruction and demonstrates how the associated statistics can provide a diagnostic tool to improve SPA. Small numbers of images already give sufficient information on micrograph quality and the amount of data required to reach high resolution. Such feedback allows the microscopist to improve sample-preparation and imaging parameters before committing to extensive data collection. Once a larger data set is available, a B factor can be determined describing the suppression of the signal owing to one or more causes, such as specimen movement, radiation damage, alignment inaccuracy and structural variation. Insight into the causes of signal suppression can then guide the user to consider appropriate actions to obtain better reconstructions.

In studying biologically relevant molecules, the ultimate aim is to visualize them in their native environment. One technique that has the potential to resolve individual molecules in cells and tissues is cryo-electron microscopy of vitreous sections (CEMOVIS) combined with tomography. We are interested in revealing the disposition of the retinal adhesion protein, retinoschisin (RS1), within the retina. Immuno-fluorescence and immuno-electron microscopy showed that RS1 is abundant in the intercellular spaces between the inner segments of photoreceptors, and between bipolar cells. Loss-of-function mutations in RS1 cause X-linked retinoschisis (XLRS), a debilitating disease associated with the formation of retinal cavities or cysts (schisis) and vision loss in young males. We solved the structure to 4.1 Å by cryo-electron microscopy (cryoEM), a 16-mer composed of two octameric rings with a total weight of ∼380 kDa. From this structure it is clear that many mutations in the subunit interfaces would preclude assembly of the rings. However, other mutation sites are exposed at the periphery and it was not evident how they would cause disease. In further cryoEM experiments, we observed that RS1 double rings self-assemble into extensive branched networks, suggesting a mechanism for its adhesive function. To ascertain whether this assembly also occurs in situ, we are examining mouse retinal tissue using CEMOVIS and cryo-electron tomography. We obtained images of 50 nm cryosections that show clear distinction of microtubular protofilaments separated by ∼5 nm. This resolution is clearly sufficient to see the RS1 double ring and whether it assembles into networks in the intercellular space between photoreceptors and bipolar cells.

Herpes simplex virus type 1 (HSV-1) contains a large double-stranded DNA genome of about 152,000 base pairs, which requires accommodation in a correspondingly massive particle.In the nucleus, viral DNA is packaged into a spherical precursor particle called the procapsid via the portal protein, pUL6, which occupies one of the procapsid's twelve vertices.Upon DNA insertion, the procapsid matures into an angular, icosahedral capsid and undergoes the subsequent steps in the viral life cycle.While the overall structure of the viral portal has been known for some time [1, 2], its exact orientation and position in the context of the capsid have been controversial.Investigations have been hampered by the fact that the one portal-bearing vertex is practically visually indistinguishable from the other eleven vertices.This has led to various publications, using different technical approaches, placing the portal both inside the particle as well as in line with other capsomers and in various orientations [3][4][5][6].

Contractile injection systems are sophisticated multiprotein nanomachines that puncture target cell membranes. Although the number of atomic-resolution insights into contractile bacteriophage tails, bacterial type six secretion systems and R-pyocins is rapidly increasing, structural information on the contraction of bacterial phage-like protein-translocation structures directed towards eukaryotic hosts is scarce. Here, we characterize the antifeeding prophage AFP from Serratia entomophila by cryoelectron microscopy. We present the high-resolution structure of the entire AFP particle in the extended state, trace 11 protein chains de novo from the apical cap to the needle tip, describe localization variants and perform specific structural comparisons with related systems. We analyse inter-subunit interactions and highlight their universal conservation within contractile injection systems while revealing the specificities of AFP. Furthermore, we provide the structure of the AFP sheath-baseplate complex in a contracted state. This study reveals atomic details of interaction networks that accompany and define the contraction mechanism of toxin-delivery tailocins, offering a comprehensive framework for understanding their mode of action and for their possible adaptation as biocontrol agents.

Replica-based freeze-fracture and freeze-etching electron microscopy methods provide surface topography information, particularly suited to studying membrane protein complexes in their native context. The fidelity and resolution of metal replicas is limited by the inherent property of metal atoms to crystallize. To overcome the limitations of metal replicas, we combined amorphous carbon replicas with phase-contrast electron microscopy. Using this approach, tight junction intramembrane fibrils were shown to have a double stranded morphology.