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

• Published in: Nanoscale Vol. 13; no. 15; pp. 7285 - 7293
• Main Authors: Casasanta, Michael A, Jonaid, G. M, Kaylor, Liam, Luqiu, William Y, Solares, Maria J, Schroen, Mariah L, Dearnaley, William J, Wilson, Jarad, Dukes, Madeline J, Kelly, Deborah F
• Format: Journal Article
• Language: English
• Published: England Royal Society of Chemistry 21.04.2021
• Subjects: Algorithms; Antibodies; Binding sites; Coronavirus Nucleocapsid Proteins - chemistry; Cryoelectron Microscopy; Macromolecules; Microscopy; Molecular structure; Molecular Weight; Pandemics; Phosphoproteins - chemistry; Protein Structure, Secondary; Proteins; SARS-CoV-2 - chemistry; Semiconductors; Severe acute respiratory syndrome coronavirus 2; Transmission electron microscopy; Viral diseases
• ISSN: 2040-3364; 2040-3372; 2040-3372
• DOI: 10.1039/d1nr00388g

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. Here we show a major technical advance to study small proteins using cryo-EM. Structural models for the SARS-CoV-2 Nucleocapsid (N) protein are presented along with other coronavirus counterparts for therapeutic development.