| The life cycle of double-stranded DNA viruses like assembly,maturation and genome ejection have long been attracting considerable attention.To elaborate on life course of the virus through their threedimensional structures captured by structural biology technique in basical research and application of life science is of great significance.The research object of this paper is the symmetry-mismatch reconstruction of phage T7 by cryo-electron microscopy with single particle technique(cryoEM).The phage T7 virus has a very large molecular weight and is difficult to grow into a three-dimensional crystal.Therefore,cryo-EM has become the most powerful means of analyzing the three-dimensional structure of the virus.However,compared with highly symmetric viral capsid proteins,the solution of non-icosahedral symmetric proteins for bacteriophage virus remains insufficient.The main reason is the information of non-icosahedral symmetric proteins is shadowed by the symmetry imposed in the analysis of icosahedral symmetric capsid proteins.In order to analyze the symmetry-mismatch reconstruction structure of phage T7,the theory of three-dimensional reconstruction of virus must be started.Traditional three-dimensional reconstruction theory of icosahedral viruses has so far been developed.In recent years,the theory and algorithmhas also been made rapid progress,especially the proposal of local reconstruction algorithm.This proposal makes it very convenient for people to study non-icosahedral symmetric proteins.In this paper,from the symmetry-mismatch algorithm perspective,we propose a symmetrymismatch reconstruction algorithm and the local reconstruction algorithm is introduced.By the end of our study,we obtained the three-dimensional structure of phage T7 in four states by symmetry-mismatch reconstruction.At the same time,we elucidate the mechanisms of phage T7 assembly and genomic release.The main tasks of this project are as follows:1)A seriers new algorithm of symmetry-mismatch reconstruction was proposed,and a large-scale computing package was written based on the new method.The new algorithm includes four parts: the icosahedral reconstruction,the initial model of asymmetric reconstruction generation,symmetric mismatch reconstruction and local reconstruction.The new algorithm and program can realize virus symmetric mismatch reconstruction from scratch.2)Phage T7 packaged samples were prepared and the data of the packaged state were obtained.This paper proposes a new algorithm of parsing the packing state symmetry-mismatch refactoring 15 ? resolution three-dimensional structure.We reconstructed the structure of core proteins and portal protein by the local reconstruction algorithm at 6.0? and 4.9 ? resolution respectively.With these high-resolution maps,we built atomic models to confirm our density in atomic scale.3)Mature phage T7 samples were prepared and mature data were obtained.This paper proposes a new algorithm of parsing the mature state of symmetry-mismatch refactoring 7.0 ? high-resolution threedimensional structure.We reconstructed the structure of the core protein,portal protein,tail proteins and tail fiber N-termini by the local reconstruction algorithm at 3.8 ? resolution.Atomic model has been built based on the map above.4)Phage T7 released state samples were prepared,and data of released state real state and empty state were obtained.This paper proposes a new algorithm of resolve the structure of release real mentality and empty states at 10 ? resolution and 15 ? resolution respectively.We reconstructed the structure of the portal protein,tail proteins and tail fiber N-termini by the local reconstruction algorithm at 4.5 ? resolution and an atomic model has been built based on the map above.5)This paper studied the assembly and DNA release mechanism of phage T7 virus particles,proposed the structure-based virus assembly and DNA release mechanism,and further improved the assembly and DNA release mechanism of phage T7 virus particles. |
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