Practical Studies On Correcting Ewald Sphere Curvature On High Resolution Cryo-em Three Dimensional Reconstruction

The high-resolution cryo electron microscopy(Cryo-EM) technology hasbeen well considered as the most vigorous mainstream experimental technology in modern structural biology. Theresearch objects rangefrommicro-order to nano-order, mainly include protein, nucleic acid, macromoloecular complex andorganelle. It hasatomic resolution analytical capacity, can be used to overcome certain technical limits brought by X-ray diffraction and nuclear magnetic resonance (NMR) techniques. However, due to the inherent characteristics of the cryo-electron microscopy single-particle technology, i. e. using low-dose electron imaging, embeding sample in amorphous ice, result in low contrastimage without sufficient signal-to-noise ratio. Hence, here weshallintroducean innovative approach carrying over and blazing new trails of the traditional method and overcome this problem.High-resolution cryo-EM technology has been widely used in the virus structure (icosahedron) elucidation, however, there exists two critical barriers:large computation of high-resolution reconstruction and the intrinsic Ewald sphere curvature, narrow the prospects of its application, especially the latter one. Sinceelectronic wavelength determines the highest attainable theoretical resolution, if the thickness of the sample exceeds the depth of field, the corresponding symmetric scatteredbeamwill no longer obey the Friedel’s law, then, the amplitude and phase of the Fourier coefficients corresponding to certainresolution would no longer follow the complex conjugate relationship. At this point, the Fourier coefficients obtained from the real imagearea linear combination of the corresponding three-dimensional structure factors, rather than complyingwith the Friedel-related underthe traditional sense. We need to recover the Fourier coefficients of the three-dimensional structure from theactual image. Under this purpose, we have tomake some improvements for current’imaging methods.By comparingwith the current published methods andtaking into account the limitations of real experiments, such as the presence of astigmatism, defocus, the actual accuracy of the magnification, through theoretical analysis and experimentaltesting, technical studies, we worked out a comprehensive imaging method to overcome the above problems. On this basis, the human cytomegalovirus (HCMV) virus is choosen asour researchsample, we got an structurewhose resolution is better than4.5A, and then start building an atomic model. Thusthis verifies the correctness and reliability of the established methodology and technique for large size particle imaging.