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

Expression of concern for 'Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy' by Michael A. Casasanta et al., Nanoscale, 2021, 13, 7285-7293, https://doi.org/10.1039/D1NR00388G.

Structural Insights of the SARS-CoV-2 Nucleocapsid Protein: Implications for the Inner-workings of Rapid Antigen Tests.

The nucleocapsid (N) protein is an abundant component of SARS-CoV-2 and a key analyte for lateral-flow rapid antigen tests. Here, we present new structural insights for the SARS-CoV-2 N protein using cryo-electron microscopy (EM) and molecular modeling tools. Epitope mapping based on structural data supported host-immune interactions in the C-terminal portion of the protein, while other regions revealed protein-protein interaction sites. Complementary modeling results suggested that N protein structures from known variants of concern (VOC) are nearly 100% conserved at specific antibody-binding sites. Collectively, these results suggest that rapid tests that target the nucleocapsid C-terminal domain should have similar accuracy across all VOCs. In addition, our combined structural modeling workflow may guide the design of immune therapies to counter viral processes as we plan for future variants and pandemics.

High-Resolution Imaging of Human Cancer Proteins Using Microprocessor Materials.

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.

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

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 Disease-Relevant p53.

The tumor suppressor protein TP53 (p53) plays a multifaceted role in all cells of the human body. Mutations in the TP53 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 p53 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.

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

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.