Cryo-electron microscopy (cryo-EM) is a powerful tool for macromolecular to near-atomic resolution structure determination in the biological sciences. The specimen is maintained in a near-native environment within a thin film of vitreous ice and imaged in a transmission electron microscope. The images can then be processed by a number of computational methods to produce three-dimensional information. Recent advances in sample preparation, imaging, and data processing have led to tremendous growth in the field of cryo-EM by providing higher resolution structures and the ability to investigate macromolecules within the context of the cell. Here, we review developments in sample preparation methods and substrates, detectors, phase plates, and cryo-correlative light and electron microscopy that have contributed to this expansion. We also have included specific biological applications.

Journal Article Navigating the Job Market for Careers Inside and Outside of Academia. Get access Benjamin Bammes, Benjamin Bammes Direct Electron, LP, San Diego, CA, USA Search for other works by this author on: Oxford Academic Google Scholar Gabriella Kiss, Gabriella Kiss FEI, Hillsboro, OR, USA Search for other works by this author on: Oxford Academic Google Scholar Paula Flicker, Paula Flicker National Institute of General Medical Sciences, National Institutes of Health, Bethesda, MD, USA Search for other works by this author on: Oxford Academic Google Scholar Scott M Stagg Scott M Stagg Institute of Molecular Biophysics, Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 22, Issue S3, 1 July 2016, Pages 2076–2077, https://doi.org/10.1017/S1431927616011223 Published: 25 July 2016

Correlative light and electron microscopy (CLEM) combines localization data from fluorescent microscopy (FM) with ultra-structural information from electron microscopy (EM). Recent improvements on fluorescent dyes and proteins enabled wider applications of this approach [1][2]. One of the main difficulties in CLEM is that often time, sample processing for EM imaging quenches the FM signal. Despite those difficulties, some newer labeling methods enable to perform CLEM experiments with a high level of success. The study of mouse hepatitis virus replication in murine cells described below demonstrates the most recent improvement made in this field.

Journal Article Zernike Phase Contrast Cryo-Electron Tomography of Bacteria and Viruses Get access ER Wright, ER Wright Emory University School of Medicine, Division of Pediatric Infectious Diseases, Children’s Healthcare of Atlanta, Atlanta, GeorgiaRobert P. Apkarian Integrated Electron Microscopy Core, Emory University, Atlanta, Georgia Search for other works by this author on: Oxford Academic Google Scholar R C Guerrero-Ferreira, R C Guerrero-Ferreira Emory University School of Medicine, Division of Pediatric Infectious Diseases, Children’s Healthcare of Atlanta, Atlanta, Georgia Search for other works by this author on: Oxford Academic Google Scholar G Kiss, G Kiss Emory University School of Medicine, Division of Pediatric Infectious Diseases, Children’s Healthcare of Atlanta, Atlanta, Georgia Search for other works by this author on: Oxford Academic Google Scholar J D Strauss, J D Strauss Emory University School of Medicine, Division of Pediatric Infectious Diseases, Children’s Healthcare of Atlanta, Atlanta, Georgia Search for other works by this author on: Oxford Academic Google Scholar C M Hampton C M Hampton Emory University School of Medicine, Division of Pediatric Infectious Diseases, Children’s Healthcare of Atlanta, Atlanta, Georgia Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 20, Issue S3, 1 August 2014, Pages 204–205, https://doi.org/10.1017/S1431927614002748 Published: 27 August 2014

ABSTRACT Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization. IMPORTANCE hRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable organizational profiles. Depending on the virus morphology examined, the amount of M ranged from 24% to 86%. We complemented the cryo-imaging studies with fluorescence microscopy, dSTORM, and a proximity ligation assay to provide additional evidence that M2-1 is incorporated into viral particles and is positioned between M and RNP. The results highlight the impact of M and M2-1 on the regulation of hRSV organization.

ABSTRACT Paramyxovirus attachment and fusion (F) envelope glycoprotein complexes mediate membrane fusion required for viral entry. The measles virus (MeV) attachment (H) protein stalk domain is thought to directly engage F for fusion promotion. However, past attempts to generate truncated, fusion-triggering-competent H-stem constructs remained fruitless. In this study, we addressed the problem by testing the hypothesis that truncated MeV H stalks may require stabilizing oligomerization tags to maintain intracellular transport competence and F-triggering activity. We engineered H-stems of different lengths with added 4-helix bundle tetramerization domains and demonstrate restored cell surface expression, efficient interaction with F, and fusion promotion activity of these constructs. The stability of the 4-helix bundle tags and the relative orientations of the helical wheels of H-stems and oligomerization tags govern the kinetics of fusion promotion, revealing a balance between H stalk conformational stability and F-triggering activity. Recombinant MeV particles expressing a bioactive H-stem construct in the place of full-length H are viable, albeit severely growth impaired. Overall, we demonstrate that the MeV H stalk represents the effector domain for MeV F triggering. Fusion promotion appears linked to the conformational flexibility of the stalk, which must be tightly regulated in viral particles to ensure efficient virus entry. While the pathways toward assembly of functional fusion complexes may differ among diverse members of the paramyxovirus family, central elements of the triggering machinery emerge as highly conserved.

Enveloped virus release is driven by poorly understood proteins that are functional analogs of the coat protein assemblies that mediate intracellular vesicle trafficking. We used differential electron density mapping to detect membrane integration by membrane-bending proteins from five virus families. This demonstrates that virus matrix proteins replace an unexpectedly large portion of the lipid content of the inner membrane face, a generalized feature likely to play a role in reshaping cellular membranes.

Electron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature. In contrast, compact M protein is associated with flexibility and low spike density. Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus assembly.