| Soft matter is a class of materials composed of macromolecules or groups that do not belong to the category of conventional fluids or solids in a simple sense,including liquid crystals,colloids,polymers,proteins,metal organic frameworks,covalent organic frameworks,etc.They not only exist commonly in people’s daily life,but also are the material bases of life,so the optimization of properties and the in-depth understanding of functional mechanisms have been the hot spots of research in related fields.Structure determines the performance and function of substance,which makes the accurate characterization of the structure of soft matter indispensable for understanding its performance and elucidating its mechanism.In recent years,cryo-electron microscopic reconstruction has become one of the important methods to study the structure of soft matter,especially those that are structurally unstable,intolerant to electron irradiation,and require an aqueous environment.In this thesis,we have conducted a systematic structural study of three representative soft matter by using cryo-electron microscopy techniques such as Microcrystal Electron Diffraction and Single Particle Analysis as the main tools for structural analysis.The results not only provide the structural basis for the degradation of Metal-Organic Framework(MOF),the vaccine development of Foot-and-Mouth Disease Virus(FMDV),and the enzymatic synthesis of t~6ATP and t~6ADP,but also further illustrate the importance of cryo-electron microscopy in"in situ"observation of samples.The unique advantages of"in situ"observation of the natural structure of samples,study of unstable transient structures,and analysis of intermolecular interactions are further illustrated.The specific research results are as follows:Zn-based MOFs are unstable in water and will collapse and degrade,while their degradation mechanism is still unclear.Using cryo-electron microscopy as a test tool,we investigated the degradation process of a typical Zn-based MOF in water.Combined with the micro crystal electron diffraction method,we not only confirmed a variety of hydrated structures identified by single-crystal X-ray diffraction,but also resolved a previously unknown intermediate state structure.The results provide a structural basis for further revealing the degradation mechanism of MOF.FMDV is a virulent pathogen that can infect even-toed ungulates,and an in-depth understanding of its antigenic epitopes is fundamental to the development of relevant vaccines.By using cryo-electron microscopic three-dimensional reconstruction technique,we analyzed the structure of FMDV serotype A in complex with neutralizing antibody P22C,and identified D72,a potential key antigen determinant,through the analysis of the structural characteristics and binding free energy of th FMDV serotype A epitope,and found that H77W and S195L mutations are expected to improve the antigenicity of FMDV,providing structural basis and clues for the design of FMDV serotype A vaccine molecules.TsaD-TsaC-SUA5-TcdA(TsaN)protein is a modular protease that is evolutionarily related to TsaC/Sua5 protein,TsaD/Kae1/Qri7 protein and E.coli TcdA,and has the catalytic function to biosynthesize various modifications such as TC-AMP,t~6A and ct~6A.Using cryo-electron microscopic three-dimensional reconstruction techniques,we resolved the dimeric structure of TsaN proteins,thus providing a structural basis for elucidating the functional mechanism of this protease.Structural analysis showed that the two TsaN proteins bind through the dimerization of the TcdA structural domain.Each TcdA structural domain is linked to the TsaD-TsaC-SUA5structural domain within the same monomer by a flexible short chain and interacts with the SUA5 structural domain of the other monomer in a limited manner,resulting in a highly flexible dimer structure.The results provide a structural basis for enzymatic synthesis of t~6ATP and t~6ADP,and the in-depth understanding of the catalytic mechanism of the conversion of t~6A to ct~6A. |
