A general and modular DARPin-apoferritin scaffold enables the visualization of small proteins by cryo-EM

Abstract

Structural biology understands and elucidates biological mechanisms by analyzing the structure of biological macromolecules. It can explore and understand the pathogenesis of diseases, as well as develop more effective treatment methods. In addition, structural biology facilities design biological antibiotics and other important biological compounds. There are three analytical methods for structural biology. Nuclear Magnetic Resonance (NMR), X-ray crystallography, and cryo-electron microscope (cryo-EM). NMR can be used to observe the dynamic process of proteins, but it is limited to stable proteins with small molecular weights. Although X-ray has analyzed a lot of very important structures, this method is also powerless for proteins that are unable to diffract. With the development of cryo-EM at present, this method has analyzed many very important membrane proteins and protein complexes, but it is difficult to achieve satisfactory resolution for proteins with small molecular weights. Many strategies have attempted to use cryo-EM to resolve small proteins, mainly divided into three categories: 1. improving cryo-EM machine components, 2. optimizing protein sample preparation and data analysis, 3. linking small proteins to large proteins to resolve the entire protein complex. The first two strategies are very demanding in the experiment and unsuitable for most proteins. The current, more effective solution is the third strategy: rigidly connect the small protein with a large scaffold protein and determine the three-dimensional (3D) structure of the small protein by solving the structure of the entire large protein complex. However, the resolution of the target protein currently solved is not satisfactory. In this study, based on the above method, suitable bases and adaptor proteins to form scaffold proteins were screened and the connection between the adaptor and base was optimized to enhance the rigidity of the connection. In addition, by screening suitable base proteins, the symmetry of the base is used to reduce the amount of electron microscopy images collected and the orientation preference of protein particles on the copper mesh to obtain a high-resolution cryo-EM structure of small proteins. Finally, a relatively high-resolution backbone protein (2.5 Å) and a high-resolution small target protein (GFP.2.8-3.8 Å) was obtained and the MBP high-resolution structure was determined by applying this method. In conclusion, the method of obtaining small proteins by cryo-EM and obtained relatively high 3D structures of small proteins was improved. In future, this method will apply to more proteins with unknown structures.