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.

Elucidating structural variability in p53 conformers using combinatorial refinement strategies and molecular dynamics.

Low molecular weight proteins and protein assemblies can now be investigated using cryo-electron microscopy (EM) as a complement to traditional structural biology techniques. It is important, however, to not lose sight of the dynamic information inherent in macromolecules that give rise to their exquisite functionality. As computational methods continue to advance the field of biomedical imaging, so must strategies to resolve the minute details of disease-related entities. Here, we employed combinatorial modeling approaches to assess flexible properties among low molecular weight proteins (~100 kDa or less). Through a blend of rigid body refinement and simulated annealing, we determined new hidden conformations for wild type p53 monomer and dimer forms. Structures for both states converged to yield new conformers, each revealing good stereochemistry and dynamic information about the protein. Based on these insights, we identified fluid parts of p53 that complement the stable central core of the protein responsible for engaging DNA. Molecular dynamics simulations corroborated the modeling results and helped pinpoint the more flexible residues in wild type p53. Overall, the new computational methods may be used to shed light on other small protein features in a vast ensemble of structural data that cannot be easily delineated by other algorithms.

Delineating Conformational Variability in Small Protein Structures Using Combinatorial Refinement Strategies.

As small protein assemblies and even small proteins are becoming more amenable to cryo-Electron Microscopy (EM) structural studies, it is important to consider the complementary dynamic information present in the data. Current computational strategies are limited in their ability to resolve minute differences among low molecular weight entities. Here, we demonstrate a new combinatorial approach to delineate flexible conformations among small proteins using real-space refinement applications. We performed a meta-analysis of structural data for the SARS CoV-2 Nucleocapsid (N) protein using a combination of rigid-body refinement and simulated annealing methods. For the N protein monomer, we determined three new flexible conformers with good stereochemistry and quantitative comparisons provided new evidence of their dynamic properties. A similar analysis performed for the N protein dimer showed only minor structural differences among the flexible models. These results suggested a more stable view of the N protein dimer than the monomer structure. Taken together, the new computational strategies can delineate conformational changes in low molecular weight proteins that may go unnoticed by conventional assessments. The results also suggest that small proteins may be further stabilized for structural studies through the use of solution components that limit the movement of external flexible regions.

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.

Complete Models of p53 Better Inform the Impact of Hotspot Mutations.

Mutations in tumor suppressor genes often lead to cancerous phenotypes. Current treatments leverage signaling pathways that are often compromised by disease-derived deficiencies in tumor suppressors. P53 falls into this category as genetic mutations lead to physical changes in the protein that impact multiple cellular pathways. Here, we show the first complete structural models of mutated p53 to reveal how hotspot mutations physically deviate from the wild-type protein. We employed a recently determined structure for the p53 monomer to map seven frequent clinical mutations using computational modeling approaches. Results showed that missense mutations often changed the conformational structure of p53 in the DNA-binding site along with its electrostatic surface charges. We posit these changes may amplify the toxic effects of these hotspot mutations by destabilizing an important zinc ion coordination region in p53 to impede proper DNA interactions. These results highlight the imperative need for new studies on patient-derived proteins that may assist in redesigning structure-informed targeted therapies.

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 is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-Å-10 Å. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

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.

Correcting errors in the BRCA1 warning system.

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, BRCA15382insC, 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 BRCA15382insC, 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.

Bifunctional Molecular Probes for Activity-Based Visualization of Quinone-Dependent Amine Oxidases.

The design, synthesis, and evaluation of two bifunctional molecular probes that can be used to visualize quinone-dependent amine oxidase enzymes in an activity-dependent manner are described. These probes use alkylhydrazines to irreversibly bind the target enzymes, which can then be visualized with either Western blotting or in-gel fluorescence. The results show that the Western blotting readout, which utilizes commercially available anti-nitrophenyl antibodies to detect a simple dinitrophenyl antigen, provides a stronger readout than the fluorescein-based fluorescence readout. This visualization strategy can be used to measure the potency of enzyme inhibitors by selectively visualizing the active enzyme that remains after treatment with an inhibitor. Looking forward, this probe molecule and visualization strategy will enable activity-based protein-profiling experiments, such as determining inhibitor selectivity values within full proteome mixtures, for this family of amine oxidase enzymes.