Near Atomic Resolution Single Particle Cryo EM Reconstruction Methodology Analysis And Application

As one of the leading subject in modern life sciences,structural biology plays a pivotal role in the study of biological phenomena and laws.Methods for structural biology analysis include X-ray crystallography,NMR and cryo electron microscopy(CryoEM),each having its own characteristics.X-ray crystallography is suitable for small proteins,that can form ordered structure into 3D crystals.Diffraction data is needed to resolve the structure of the protein.NMR spectroscopy can determine protein structures in different conformations in solution,and can be used in the study of molecular flexibility,molecular interactions and molecular dynamics but with very strict limitations of molecular weight.Only preoteins with low molecular weight(<40-50 kDa)are suitable for NMR studies.In comparision,CryoEM does not require protein crystallization,and is suitable for larger molecular weight proteins,thus becoming the first choice for the study of biological macromolecular structure.The emergence of direct electron detector and development of algorithm for motion correction and classification make CryoEM field enter into the rapid development period.Most protein complexes with relatively high molecular weight could be solved to near atomic resolution using CryoEM.CryoEM has become one of the most important research tools in structural biology.Although analytical high-resolution CryoEM structure has become possible,there are still many problems,such as how to perform structural analysis,how to optimize the reconstruction strategies,to be solved.For different projects,different reconstruction strategies will be used,and the most effecient one will be acquired through trial and error.In this thesis,I established and improved the single particle analysis procedure aiming for high resolution single particle CryoEM,especially the helical single particle analysis.First,we have a brief introduction for the electron microscope image formation and three dimensional reconstruction principles.On the bases of these introduction,the human T4-?-secretase and the helical filaments formed by human Rad51 and DNA were chosen as examples to elaborate how to do the high resolution structure determination and analysis,during which several attempts have been made and finally provided near atomicresolution structures.Based on the high-resolution structure,atomic model building is possible and structural analysis could be done to provide structural basis for their biological function and also provide basis for their relevant biological questions.In the human T4-?-secretase project,we solved a Cryo EM map at about 4.32 ? resolution,which reveals the assembly mechanism of human ?-secretase for the four components.Our EM map also provides structural basis for the functional and structural analysis for ?-secretase in the future.In the Rad51 project,we got both pre-synaptic and post-synaptic complex in high-resolution and an intermediate state in synaptic complex in medium resolution,which may serve as structure foundation for the Rad51-mediated DNA repair.High-resolution structure analysis method should base on sample characteristics and achieve the optimal one through trial and error,indicating high-resolution cryo-EM structure still needs to resolve methodological exploration,which has significance in high-resolution structure determination.