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A collaborative team from the Rosalind Franklin Institute, the University of Oxford, and Diamond Light Source has developed a breakthrough method that makes it possible to image very small proteins using cryo-electron microscopy (cryo-EM). The results are published in .

By designing new bifunctional, bispecific nanobody scaffolds, the researchers have overcome one of the biggest challenges in the field: visualizing proteins under 50 kDa in size. Using this approach, the team solved the structure of hen egg white lysozyme—at just 14 kDa, the smallest protein ever resolved by cryo-EM.

This advance is highly significant, as nearly 75% of human protein-coding genes produce proteins within this small size range. Many of these proteins are critical to cell function and play key roles in health and disease.

In recent years, single-particle cryo-EM has become a mainstream method for determining protein structures. One of the technique's main advantages is the ability to show the molecular details of structures in a near native state.

However, imaging small proteins has remained challenging due to their low signal-to-noise ratio, which leads to difficulty during data processing with particle picking and alignment, and ultimately historically leading to low resolution reconstructions.

The new nanobody scaffold overcomes these data processing challenges by bonding the small proteins to the ends of bifunctional, bispecific nanobodies, which increases apparent size and gives them a distinct geometry.

"This has been the biggest challenge I've ever faced, and it has also been a valuable learning experience on my path to becoming an independent researcher. This new technique has immense potential to become a universally applicable tool—not only in structural research but across many other fields, reasoned by its bispecific capabilities," says co-author Gangshun Yi, eBIC postdoctoral research assistant.

Dimitrios Mamalis, a joint Ph.D. student between the University of Oxford and the Franklin, explains how this work got started: "It started as a Friday afternoon experiment, which means that it wasn't the main focus of our research at the time, but it has ended up in a great result.

"Mingda Ye was originally working on gembodies for crystallography and then we wondered if we could transfer this method to cryo-EM if we could conjugate the gembodies in solution. This was when I started to find the method and optimize the reactions and conditions, linking up with Gangshun Yi to get the structures solved."

Unlike many similar approaches, the method is modular and does not require laborious re-optimization for each new protein target. It also allows two proteins to be studied simultaneously, even if they are of different sizes, by attaching them to opposite ends of the scaffold.

Professor Ben Davis, science director at the Franklin and co-author of the study, highlighted the power of this collaborative work: "This was a wonderful, organic collaboration that grew out of parallels between crystallization and covalent trapping of proteins in solution. The sidechain-to-sidechain conjugation worked remarkably well, and the efficiency of the method is striking. It's an exciting and pragmatic new way to study proteins."