advancing-high-resolution-imaging-virus-assemblies-liquid

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

Pennsylvania State University

Given its important role in human health and disease, remarkably little is known about the full-length three-dimensional (3D) molecular architecture of the breast cancer type 1 susceptibility protein (BRCA1), or its mechanisms to engage the tumor suppressor, TP53 (p53). Here, we show how a prevalent cancer-related mutation in the C-terminal region of the full-length protein, BRCA1, affects its structural properties, yet can be biochemically corrected to restore its functional capacity. As a downstream consequence of restoring the ubiquitin ligase activity of mutated BRCA1, the DNA repair response of p53 was enhanced in cellular extracts naturally deficient in BRCA1 protein expression. Complementary structural insights of p53 tetramers bound to DNA in different stage of the repair process support these biochemical findings in the context of human cancer cells. Equally important, we show how this knowledge can be used to lower the viability of breast cancer cells by modulating the stability of the BRCA1 protein and its associated players.

Correcting errors in the BRCA1 warning system.

The fight against human disease requires a multidisciplinary scientific approach. Applying tools from seemingly unrelated areas, such as materials science and molecular biology, researchers can overcome long-standing challenges to improve knowledge of molecular pathologies. Here, custom-designed substrates composed of silicon nitride (SiN) are used to study the 3D attributes of tumor suppressor proteins that function in DNA repair events. New on-chip preparation strategies enable the isolation of native protein complexes from human cancer cells. Combined techniques of cryo-electron microscopy (EM) and molecular modeling reveal a new modified form of the p53 tumor suppressor present in aggressive glioblastoma multiforme cancer cells. Taken together, the findings provide a radical new design for cryo-EM substrates to evaluate the structures of disease-related macromolecules.

Cryo-EM-On-a-Chip: Custom-Designed Substrates for the 3D Analysis of Macromolecules.

Liquid-cell electron microscopy is a rapidly growing field in the imaging domain. While real-time observations are readily available to analyze materials and biological systems, these measurementshave been limited to the two-dimensional (2-D) image plane. Here, we introduce an exciting technical advance to image materials in 3-D while enclosed in liquid. The development of liquid-cell electron tomography permitted us to observe and quantify host-pathogen interactions in solution while contained in the vacuum system of the electron microscope. In doing so, we demonstrate new insights for the rules of engagement involving a unique bacteriophage and its host bacterium. A deeper analysis of the genetic content of the phage pathogens revealed structural features of the infectious units while introducing a new paradigm for host interactions. Overall, we demonstrate a technological opportunity to elevate research efforts for  imaging while providing a new level of dimensionality beyond the current state of the field.

Liquid-Cell Electron Tomography of Biological Systems.

Rotavirus is a well-studied RNA virus that causes severe gastroenteritis in children. During viral entry, the outer layer of the virion is shed, creating a double-layered particle (DLP) that is competent to perform viral transcription (i.e., mRNA synthesis) and launch infection. While inactive forms of rotavirus DLPs have been structurally characterized in detail, information about the transcriptionally-active DLP remains limited. Here, we used cryo-Electron Microscopy (cryo-EM) and 3D image reconstructions to compare the structures of internal protein components in transcriptionally-active versus inactive DLPs. Our findings showed that transcriptionally-active DLPs gained internal order as mRNA synthesis unfolded, while inactive DLPs remained dynamically disordered. Regions of viral protein/RNA constituents were analyzed across two different axes of symmetry to provide a more comprehensive view of moving components. Taken together, our results bring forth a new view of active DLPs, which may enable future pharmacological strategies aimed at obliterating rotavirus transcription as a therapeutic approach.

Cryo-EM Reveals Architectural Diversity in Active Rotavirus Particles.

The tumor suppressor protein TP53 (p53) plays a multifaceted role in all cells of the human body. Mutations in the  gene are often involved in cancer induction and disease progression. Despite its important role in health and development, structural information for p53 remains incomplete. Here, we present a microchip-based technology to facilitate structural studies of p53 assemblies derived from human cancer cells. These devices do not introduce foreign sequences to the  gene and maintain naturally occurring post-translational modifications. Using cryo-electron microscopy, structures for the p53 monomer (∼50 kDa) and tetramer (∼200 kDa) were resolved to ∼4.8 and ∼7 Å, respectively. These structures revealed new insights for flexible regions of p53 along with biologically relevant ubiquitination sites. Collectively, the convergence of nanotechnology tools and structural imaging builds a strong framework to understand the oncogenic impact of p53 in human tissues.

Microchip-Based Structure Determination of Disease-Relevant p53.

Interest in cryo-Electron Microscopy (EM) imaging has skyrocketed in recent years due to its pristine views of macromolecules and materials. As advances in instrumentation and computing algorithms spurred this progress, there is renewed focus to address specimen-related challenges. Here we contribute a microchip-based toolkit to perform complementary structural and biochemical analysis on low-molecular weight proteins. As a model system, we used the SARS-CoV-2 nucleocapsid (N) protein (48 kDa) due to its stability and important role in therapeutic development. Cryo-EM structures of the N protein monomer revealed a flexible N-terminal "top hat" motif and a helical-rich C-terminal domain. To complement our structural findings, we engineered microchip-based immunoprecipitation assays that led to the discovery of the first antibody binding site on the N protein. The data also facilitated molecular modeling of a variety of pandemic and common cold-related coronavirus proteins. Such insights may guide future pandemic-preparedness protocols through immuno-engineering strategies to mitigate viral outbreaks.

Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy.

Liquid-phase electron microscopy (LP-EM) is an exciting new area in the materials imaging field, providing unprecedented views of molecular processes. Time-resolved insights from LP-EM studies are a strong complement to the remarkable results achievable with other high-resolution techniques. Here, the opportunities to expand LP-EM technology beyond 2D temporal assessments and into the 3D regime are described. The results show new structures and dynamic insights of human viruses contained in minute volumes of liquid while acquired in a rapid timeframe. To develop this strategy, adeno-associated virus (AAV) is used as a model system. AAV is a well-known gene therapy vehicle with current applications involving drug delivery and vaccine development for COVID-19. Improving the understanding of the physical properties of biological entities in a liquid state, as maintained in the human body, has broad societal implications for human health and disease.

High-Resolution Imaging of Human Viruses in Liquid Droplets.

Liquid-electron microscopy (EM), the room-temperature correlate to cryo-EM, is a rapidly growing field providing high-resolution insights of macromolecules in solution. Here, we describe how liquid-EM experiments can incorporate automated tools to propel the field to new heights. We demonstrate fresh workflows for specimen preparation, data collection, and computing processes to assess biological structures in liquid. Adeno-associated virus (AAV) and the SARS-CoV-2 nucleocapsid (N) were used as model systems to highlight the technical advances. These complexes were selected based on their major differences in size and natural symmetry. AAV is a highly symmetric, icosahedral assembly with a particle diameter of ~25 nm. At the other end of the spectrum, N protein is an asymmetric monomer or dimer with dimensions of approximately 5–7 nm, depending upon its oligomerization state. Equally important, both AAV and N protein are popular subjects in biomedical research due to their high value in vaccine development and therapeutic efforts against COVID-19. Overall, we demonstrate how automated practices in liquid-EM can be used to decode molecules of interest for human health and disease.

Automated Tools to Advance High-Resolution Imaging in Liquid.

Mutations in tumor suppressor genes, such as Tumor Protein 53 (TP53), are heavily implicated in aggressive cancers giving rise to gain- and loss-of-function phenotypes. While individual domains of the p53 protein have been studied extensively, structural information for full-length p53 remains incomplete. Functionalized microprocessor chips (microchips) with properties amenable to electron microscopy permitted us to visualize complete p53 assemblies for the first time. The new structures revealed p53 in an inactive dimeric state independent of DNA binding. Residues located at the protein-protein interface corresponded with modification sites in cancer-related hot spots. Changes in these regions may amplify the toxic effects of clinical mutations. Taken together, these results contribute advances in technology and imaging approaches to decode native protein models in different states of activation.

Article	Year
Correcting errors in the BRCA1 warning system. DNA repair\| PubMed ID: 30503669	2019
Cryo-EM-On-a-Chip: Custom-Designed Substrates for the 3D Analysis of Macromolecules. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 30963664	2019
Liquid-Cell Electron Tomography of Biological Systems. Nano letters\| PubMed ID: 31244227	2019
Cryo-EM Reveals Architectural Diversity in Active Rotavirus Particles. Computational and structural biotechnology journal\| PubMed ID: 31467657	2019
Microchip-Based Structure Determination of Disease-Relevant p53. Analytical chemistry\| PubMed ID: 33124814	2020
Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy. Nanoscale\| PubMed ID: 33889923	2021
High-Resolution Imaging of Human Viruses in Liquid Droplets. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 34302401	2021
Automated Tools to Advance High-Resolution Imaging in Liquid. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada\| PubMed ID: 35048845	2022
High-Resolution Imaging of Human Cancer Proteins Using Microprocessor Materials. Chembiochem : a European journal of chemical biology\| PubMed ID: 35789183	2022

William J. Dearnaley

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