Single-particle cryo-electron microscopy (cryo-EM) has gradually become the mainstream in the field of structure biology providing three-dimensional (3D) structural information of biological macromolecules. Technological advances promoted the emergence of atomic-resolution 3D reconstruction with more chemical details. However, sample preparation of single-particle cryo-EM is still the bottleneck for structure analysis. Due to the adsorption of air-water interface (AWI), a large proportion of protein particles adopt a preferred orientation and even partially denatured. It makes the determination of a high-resolution structure more difficult as enough amount of randomly oriented, well-structured particles is compulsory. Here I developed a new method using metallo-supramolecular branched p...[ Read more ]

Single-particle cryo-electron microscopy (cryo-EM) has gradually become the mainstream in the field of structure biology providing three-dimensional (3D) structural information of biological macromolecules. Technological advances promoted the emergence of atomic-resolution 3D reconstruction with more chemical details. However, sample preparation of single-particle cryo-EM is still the bottleneck for structure analysis. Due to the adsorption of air-water interface (AWI), a large proportion of protein particles adopt a preferred orientation and even partially denatured. It makes the determination of a high-resolution structure more difficult as enough amount of randomly oriented, well-structured particles is compulsory. Here I developed a new method using metallo-supramolecular branched polymer (MSBP) in sample preparation process to protect the protein particles away from air-water interface. The effects of application were evaluated from three views including biocompatibility, particle distribution and particle orientation by single particle cryo-EM and cryo-electron tomography (cryo-ET) techniques. High-resolution 3D reconstruction of apoferritin with MSBP demonstrated the biocompatibility and practicability. Distribution of apoferritin particles in vitreous ice layer was visualized by cryo-ET technique while orientation of haemagglutinin (HA) trimer was revealed by single particle cryo-EM technique. In short, MSBP can benefit the cryo-specimen preparation for single-particle cryo-EM in a simple and promising way.