Abstract Understanding the molecular evolution of the SARS‐CoV‐2 virus as it continues to spread in communities around the globe is important for mitigation and future pandemic preparedness. Three‐dimensional structures of SARS‐CoV‐2 proteins and those of other coronavirusess archived in the Protein Data Bank were used to analyze viral proteome evolution during the first 6 months of the COVID‐19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in &gt;48 000 viral isolates revealed how each one of 29 viral proteins have undergone amino acid changes. Catalytic residues in active sites and binding residues in protein–protein interfaces showed modest, but significant, numbers of substitutions, highlighting the mutational robustness of the viral proteome. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi‐Gaussian distribution. Detailed results are presented for potential drug discovery targets and the four structural proteins that comprise the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and protein–protein and protein–nucleic acid interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure‐based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

Evolution of the <scp>SARS‐CoV</scp>‐2 proteome in three dimensions (3D) during the first 6 months of the <scp>COVID</scp>‐19 pandemic

Institute for Quantitative Biomedicine, Rutgers, The State University of New Jersey  Piscataway New Jersey USA

Department of Chemistry and Chemical Biology Rutgers, The State University of New Jersey  Piscataway New Jersey USA

Research Collaboratory for Structural Bioinformatics Protein Data Bank Rutgers, The State University of New Jersey  Piscataway New Jersey USA

Rutgers Cancer Institute of New Jersey, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey  New Brunswick New Jersey USA

The Scripps Research Institute  La Jolla California USA

Grinnell College  Grinnell Iowa USA

University of Notre Dame  Notre Dame Indiana USA

University of Maryland Baltimore County  Baltimore Maryland USA

Stevens Institute of Technology  Hoboken New Jersey USA

Frostburg State University  Frostburg Maryland USA

Youngstown State University  Youngstown Ohio USA

University of Central Florida  Orlando Florida USA

New York City College of Technology  Brooklyn New York USA

Howard University  Washington District of Columbia USA

Watchung Hills Regional High School  Warren New Jersey USA

Xavier University  Cincinnati Ohio USA

Hope College  Holland Michigan USA

Ursinus College  Collegeville Pennsylvania USA

SUNY Oswego  Oswego New York USA

Roger Williams University  Bristol Rhode Island USA

Brandeis University  Waltham Massachusetts USA

University of Puerto Rico‐Rio Piedras  San Juan Puerto Rico

John Jay College  New York New York USA

Grand View University  Des Moines Iowa USA

Rochester Institute of Technology  Rochester New York USA

Department of Biochemistry and Microbiology Rutgers, The State University of New Jersey  New Brunswick New Jersey USA

Department of Ecology, Evolution and Natural Resources, School of Environmental and Biological Sciences Rutgers, The State University of New Jersey  New Brunswick New Jersey USA

Department of Genetics Rutgers, The State University of New Jersey, and Human Genetics Institute of New Jersey  Piscataway New Jersey USA

Center for Advanced Biotechnology and Medicine Rutgers, The State University of New Jersey  Piscataway New Jersey USA

Center for Computational and Integrative Biology Rutgers, The State University of New Jersey  Camden New Jersey USA

Department of Physics Rutgers, The State University of New Jersey  Camden New Jersey USA

Research Collaboratory for Structural Bioinformatics Protein Data Bank, San Diego Supercomputer Center University of California  San Diego, La Jolla California USA

Evolution of the SARS‐CoV‐2 proteome in three dimensions (3D) during the first 6 months of the COVID‐19 pandemic