Bacteriophages, or phages for short, are the most abundant biological entity in nature. They shape bacterial communities and are a major driving force in bacterial evolution. Their ubiquitous... Show moreBacteriophages, or phages for short, are the most abundant biological entity in nature. They shape bacterial communities and are a major driving force in bacterial evolution. Their ubiquitous nature and their potential use in medical and industrial applications make them attractive targets for fundamental and applied scientific studies. Understanding their structure and function at the molecular level is essential for understanding phage life cycles. In this thesis, I applied different cryo-EM techniques combined with advanced image processing and artificial intelligence methods to gain insight into structure and function of two bacteriophages. In both cases, these phages contain flexible elements which are essential for the infection process. While biologically highly interesting, these flexible components are especially challenging for structural studies. With the advances in computer technology and electron microscopy, researchers can now use various research methods to study different proteins and the structure and function of biological macromolecular machines. The studies presented in this thesis provide valuable insights into phages with flexible components, and provide a useful workflow for researchers with similar research topics. Show less
This thesis investigates the characteristics and the mechanisms of DNA replicative proteins through the use of biochemistry, single molecule light mycroscopy and Cryo-EM.
This thesis is concerned with the constitution of chiral responsive matrix assemblies that may undergo light-driven conformational changes and self-select vibrations to develop vibronic states for... Show moreThis thesis is concerned with the constitution of chiral responsive matrix assemblies that may undergo light-driven conformational changes and self-select vibrations to develop vibronic states for driving semi-classical coherent transfer with nearly 100% yield in a lossless NCAP process. In this work, a truncated CsmA derived from the CsmA protein from the baseplate of the Chlorobaculum tepidum has been used to engineer a chiral matrix around a zinc protoporphyrin IX (ZnPP) chromophore (Chapter 2). After the study of the specific structure of tCsmA-ZnPP complex, the mechanism of self-assembly was resolved (Chapter 3 and Chapter 4). Expression and purification of tCsmA in E.coli was explored to pave the way for other methods, such as NMR (Chapter 5). Show less
Biological molecules, especially the proteins, have a special and important function. We study their structure to understand their functions, and further make application, like the medical... Show moreBiological molecules, especially the proteins, have a special and important function. We study their structure to understand their functions, and further make application, like the medical research. The routine method is diffraction, but not work for molecules which cannot grow into crystal and molecules which their crystal are too small. Cryo-EM technique provides another way to solve their structures through their images, it does not need crystals. Meanwhile, electron diffraction can work small crystals (micro- and nano-crystals) after the sample preparation was improved. Hence, we try to build a method that can restore the structure from the crystal’s image. We collect images of protein nano-crystal, and these images were processed to enhance their contrast. The key step is to find the orientations of these images in the procedure of reconstruction, therefore, we create a method that calculates these orientations. Nano-crystals, which cannot be used in diffraction method, then can be used in this method. Show less
With the help of modern techniques of imaging processing and computing, image data obtained by electron cryo-microscopy of biomolecules can be reconstructed to three-dimensional biological models... Show moreWith the help of modern techniques of imaging processing and computing, image data obtained by electron cryo-microscopy of biomolecules can be reconstructed to three-dimensional biological models at sub-nanometer resolution. These models allow answering urgent problems in life science, for instance, the pathways directing the self-recovery system of cell, which certainly has great significance for all our lives. To determine these models, there are two main electron microscopic methods available, corresponding to its two main modes of operation: 3DEM single particle reconstruction and electron diffraction. This thesis focuses on the research and methods of 3DEM and electron diffraction, and its practical application in solving the structure of a 50S ribosomal complex, which clarifies the mechanism of cell recovery in heat shock stress. Preliminary research on a novel structure determination method by using nano-crystals resulted in a novel software suite __ EDiff __ which is a program for unit cell parameter determination, indexing and so on. Show less
