Yuqi Qin¹ and Shangyu Dang¹

¹Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

Cryo-electron microscopy (cryo-EM) has been a game-changer in the field of structural biology, giving us a closer look at the atomic structures of biological macromolecules, paving the way for groundbreaking discoveries and advancing our understanding of life itself.

However, as with any cutting-edge technology, cryo-EM comes with its fair share of challenges. One of the major hurdles lies in preparing the high-quality cryo-samples. It has been observed that approximately 90% of the sample particles are adsorbed to the air-water interface (AWI)¹. This unfortunate phenomenon leads to undesired effects like preferred orientations, aggregation, disassembly, and even denaturation of the proteins, ultimately hindering high-resolution structural determination. To overcome these challenges, various approaches have been explored, including the use of detergents, coating grids with graphene materials², employing rapid plunge-freezing robots³, and tilted image collection. While these methods have shown promise, they often require specialized equipment or complex techniques, limiting their widespread application and accessibility.

This is where our research comes into play! We've been working on a novel and straightforward technique that utilizes Metallo-Supramolecular Branched Polymer (MSBP) in cryo-sample preparation. So, what exactly is MSBP? It is a supramolecular polymer formed by functionalizing the ends of polyethylene glycol (PEG) chains with bispyridyl ligands and introducing palladium ions (Pd2+). The MSBP works its magic by keeping particles away from the AWI and promoting a more uniform distribution within the vitreous ice layer. This has been verified through single-particle cryo-EM and cryo-electron tomography analysis.

First and foremost, we have demonstrated that the introduction of MSBP in cryo-sample preparation does not affect high-resolution structure determination by comparing the cryo-EM structures of proteins samples with and without MSBP. Surprisingly, apoferritin with MSBP achieved an impressive resolution of 2.16 Å, approaching the Nyquist limit and slightly surpassing apoferritin without MSBP. Further systematic analysis revealed that MSBP pushed most particles into the central layer of the vitreous ice, leading to a significant resolution improvement through CTF refinement. The results showed that MSBP had no adverse effects on the protein structure and, in some cases, even enhanced the ice quality and resolution.

In addition, we proved that the influence of MSBP on resolution can be neglected for smaller proteins. We analyzed catalase datasets with and without MSPB and observed an acceptable gradual decrease in resolution when reconstructing maps for catalase trimers, dimers, and monomers using the same number of particles. But don't worry! To compensate for the potential resolution drop of smaller proteins caused by MSBP, we increased particle numbers by performing symmetry expansion and particle subtraction, resulting in improved resolution for catalase monomers. These findings indicate that the background noise introduced by MSBP is negligible, and potential resolution loss can be mitigated by collecting a larger dataset if needed.

But wait, there’s more! We delved deeper into the effects of MSBP by employing cryo-ET to investigate particle distribution in vitreous ice. We tested apoferritin, HA-trimer, catalase, and β-galactosidase, two membrane proteins, and CSW complex. Without MSBP, particles tended to accumulate at the air-water surface, whereas with MSBP, we observed a more uniform distribution throughout the ice. This indicates that MSBP has the potential to enhance particle distribution and alleviate issues associated with the air-water interface.

Another exciting discovery is that MSBP can tackle the issue of preferred orientation caused by AWI. The cryo-EM density map of hemagglutinin (HA) trimer exhibited distorted features when using traditional cryo-sample preparation, indicating severe preferred orientation issues. In contrast, when MSBP was introduced, the cryo-EM map exhibited higher resolution and matched the known structure of the protein. Both the 2D class average and the directional FSC indicated a more uniform angular distribution of HA-trimer particles with MSBP. Similar improvements in particle distribution were observed for catalase and β-galactosidase. This exciting discovery opens new doors for proteins with preferred orientation problems.

In summary, we have demonstrated that the incorporation of MSBP in cryo-sample preparation does not negatively impact protein resolution. Furthermore, MSBP helps keep particles away from the AWI, consequently, mitigates issues associated with AWI, such as preferred orientation problem. The simplicity and ease of implementation of MSBP make it accessible to the wider scientific community. As we look to the future, the application of MSBP in cryo-sample preparation will eventually benefit the field of cryo-EM.

Our lab is located at the Hong Kong University of Science and Technology (HKUST). We actively develop new methods in the fields of single-particle cryo-EM and cryo-ET. We are also interested in the molecular mechanisms of biological macromolecules, with a specific focus on membrane proteins and protein-DNA complexes. We welcome collaboration and invite others to join us! For more information, please visit our lab’s website: https://dang-lab.ust.hk.

Reference:

1. Noble, A. J. et al. Routine single particle CryoEM sample and grid characterization by tomography. Elife 7, e34257 (2018).
2. Wang, F. et al. Amino and PEG-amino graphene oxide grids enrich and protect samples for high-resolution single particle cryo-electron microscopy. J. Struct. Biol. 209, 107437 (2020).
3. Jain, T., Sheehan, P., Crum, J., Carragher, B. & Potter, C. S. Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. J. Struct. Biol. 179, 68–75 (2012).