# Bridging the Structural Gap: A Methodological Review of Cryo-Electron Microscopy for Underrepresented Viruses

## TL;DR

This paper reviews cryo-EM methods to study underrepresented viruses, highlighting technical challenges and solutions for better structural analysis.

## Contribution

The paper provides a methodological review and proposes strategies to address structural biases in cryo-EM for underrepresented viruses.

## Key findings

- Vertebrate viruses dominate cryo-EM structural data, while plant and insect viruses are underrepresented.
- Specialized methods like block-based local refinement and cryo-electron tomography address technical barriers in virus structure determination.
- Strategies are proposed to expand structural investigations across underrepresented host systems.

## Abstract

Cryo-electron microscopy (cryo-EM) has revolutionized structural virology, enabling routine structure determination at 2–4 Å resolution, with exceptional cases reaching 1.56 Å. The structural diversity of viruses across vertebrate, plant, and insect hosts provides fundamental insights into infection mechanisms, host–pathogen coevolution, and therapeutic target identification. However, analysis of Electron Microscopy Data Bank entries reveals notable disparities in structural coverage: among 11,717 eukaryotic virus structures (excluding bacteriophages), vertebrate viruses constitute 97.6% (n = 11,432) of deposited entries, while plant viruses (1.0%; n = 117) and insect viruses (1.4%; n = 168) remain significantly underrepresented. This bias stems from distinct technical barriers including size limitations for giant viruses exceeding 200 nm, the loss of asymmetric information during symmetry-imposed processing, and the morphological complexity of filamentous and pleomorphic viruses. Each barrier has driven the development of specialized methodological solutions: block-based local refinement overcomes through-focus variations in giant viruses, cryo-electron tomography (cryo-ET) validates and reveals asymmetric features lost in symmetrized reconstructions, and subtomogram averaging enables structural analysis of pleomorphic assemblies. This review synthesizes recent methodological advances, critically evaluates their capacity to address specific technical barriers, and proposes strategies for expanding structural investigations across underrepresented host systems to achieve comprehensive understanding of viral structural biology.