The Studies Of Symmetry-mismatch Reconstruction Of Icosahedral Viruses By Cryo-electron Microscopy And Its Applications

"Structure determines function",and it is especially for macromoleculer.Virus,a special type of macromolecular,is of vital significance of understanding pathogenesis of viral diseases,diseases contral and prevention and drug design for its structural analysis.In recent years,rapid development has taken place in Cryo-EM techenology and made it one of the most powerful analysis instrument for macromolecular structure.Moreover,as for viral structure analysis,a detailed work on such molecular weight,single-particle analysis of Cryo-EM has become the only way out.Most of viral particles have a particular structure assembled by matters of symmetrical inconformity includingis composed of an icosahedral capsid,non-icosahedral genome and messive associatedion proteins within its capsid which is called symmetry-mismatch structure.3D reconstruction of icosahedral virus has always taken the leading position in high-resolution structure analysis of Cryo-EM due to high symmetry and large molecular weight of icosahedral virus and many of new methords and technologies are realized by means of it.We can hardly lose sight of adenovirus,the first cryo-sample;HBV,the earliest sub-nanometer resolution structure;Infections ?15 virus,the oldest mainchain model;GCRV together with adenovirus,the original full atomic model.In present,with the further development of either hardware or software,near-atomic resolution 3D structure of a icosahedral virus and its full atomic model can be acquired rather easier by using single-particle analysis technology of Cryo-EM.However,the information without icosahedral symmetry inside viral capsid will,to a great extent,be discarded while reconstruction of icosahedral symmetry is being performed.Thus,structures within viral particle still remains to be explored dispite of mature development 3D reconstruction technology on icosahedral viral capsid of Cryo-EM and huge quantities of viral capsid structures solved through such methord and makes it a choke point in structure analysis of Cryo-EM.This thesis is aimed to develop symmetry-mismatch reconstruction methord and software of icosahedral viruses by cryo-electron microscopy and apply it to analysis of structure inside viral capsid hoping to break the choke point in symmetry-mismatch structure analysis of Cryo-EM.Our major works will be listed as follow:Firstly,researches of symmetry-mismatch reconstruction algorithm of icosahedral viruses by Cryo-EM.Basing on contrast separation of high-resolution image of Cryo-EM,we improved and complete the 3D reconstruction methord for symmetry-mismatch of icosahedral virus.First of all,traditional 3D reconstruction methord for icosahedral virus has been utilized to obtain icosahedral orientations of 2D images of every viral particle and high-resolution 3D structure of viral capsid.After projections corresponding to each raw particle image have been made along their particular icosahedral orientations,capsid information were subtracted from raw particle image by making comparation between their projections and themselves to extract non-icosahedral information of them.In the end,refinement was performed according to geometrical relationship of symmetry-mismatch to determine orientations and center,and unabridged symmetry-mismatch structure were obtained through non-symmetric reconstruction right behind whose resolution could be pushed up by iterationSecondly,software development and application of symmetry-mismatch reconstruction algorithm of icosahedral viruses by Cryo-EM.In this article,we present a program wrote by FROTRA.N 90 pointing at symmetry-mismatch reconstruction algorithm of icosahedral viruses by Cryo-EM and compiled in the Microsoft Windows environment.In addition,for the purpose of promoting the computation efficiency of our programs,some auxiliary programs for symmetry mismatch reconstruction have been designed and they seem to apparently work.The software is mainly comprised of three modules:1)extraction of non-icosahedral information;2)generating of initial non-icosahedral model;3)symmetry mismatch orientation determination and reconstruction.Symmetry mismatch reconstruction has been performed on both non-transeribing and transcribing cypovirus.The capsid resolution of non-transeribing CPV reaches a 3.2A resolution while its RdRp reaches a resolution of 3.9A.As for transcribing one,the resolution should be 3.2A and 3.9A for capsid and RdRp respectively.This high-resolution result can be served as a valid evidence for the efficiency of our algorithm.Thereby,conformational variation in two subdomains of RdRp inside Cypovirus(CPV)has been discovered to describe the possibility that switch-like conformational variation of RdRp from non-transcribing to transcribing state can promote despiralization course of genomic dsRNA.Thirdly,to promot the calculative accuracy of parameters for the orientation and center of virus,discussions have been made on particle extraction during symmetry mismatch reconstruction,and then a threshold value determination method for phase residual basing on statistics and a correlation parameter determination method for matching between projection and model have been put forward.A result shows that precise determination of threshold value is quite efficacious on high-resolution 3D reconstruction.