Significant advances in the past decade have enabled high-resolution structure determination of a vast variety of proteins by cryogenic electron microscopy single particle analysis. Despite improved sample preparation, next-generation imaging hardware, and advanced single particle analysis algorithms, small proteins remain elusive for reconstruction due to low signal-to-noise and lack of distinctive structural features. Multiple efforts have therefore been directed at the development of size-increase techniques for small proteins. Here we review the latest methods for increasing effective molecular weight of proteins <100 ​kDa through target protein binding or target protein fusion - specifically by using nanobody-based assemblies, fusion tags, and symmetric scaffolds. Finally, we summarize these state-of-the-art techniques into a decision-tree to facilitate the design of tailored future approaches, and thus for further exploration of ever-smaller proteins that make up the largest part of the human genome.

With severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as an emergent human virus since December 2019, the world population is susceptible to coronavirus disease 2019 (COVID-19). SARS-CoV-2 has higher transmissibility than the previous coronaviruses, associated by the ribonucleic acid (RNA) virus nature with high mutation rate, caused SARS-CoV-2 variants to arise while circulating worldwide. Neutralizing antibodies are identified as immediate and direct-acting therapeutic against COVID-19. Single-domain antibodies (sdAbs), as small biomolecules with non-complex structure and intrinsic stability, can acquire antigen-binding capabilities comparable to conventional antibodies, which serve as an attractive neutralizing solution. SARS-CoV-2 spike protein attaches to human angiotensin-converting enzyme 2 (ACE2) receptor on lung epithelial cells to initiate viral infection, serves as potential therapeutic target. sdAbs have shown broad neutralization towards SARS-CoV-2 with various mutations, effectively stop and prevent infection while efficiently block mutational escape. In addition, sdAbs can be developed into multivalent antibodies or inhaled biotherapeutics against COVID-19.