![]() ![]() Because of strong interaction between electrons and specimen, the ratio among these three types of outgoing electrons greatly depends on sample dimensions and its elemental composition. When electrons penetrate through a thin frozen-hydrated biological sample, three types of outgoing electrons reach the detector: unscattered which electrons do not make any atomic interaction, elastically and inelastically scattered by atoms in a sample during transmission. ![]() Cryo-EM data acquisition and radiation damage Full understanding of SPA and MicroED in data acquisition and analysis will facilitate radiation damage assessment and provide solution to minimize such effect for all cryo-EM applications. However, there is a significant discrepancy in rate and severity of resolution decay between the two methods. Both methods exhibited similar side chain density degeneration caused by inelastic electron scattering. Recently, SPA and MicroED were employed to assess radiation damage at high resolution. After beam-induced motion becomes correctable, radiation damage in macromolecules has become the primary outstanding resolution limit factor in all cryo-EM applications. The other new powerful tool developed in parallel to SPA is sub-micrometer 3D crystal electron diffraction (MicroED) as a part of 3D electron crystallography that allows 3D protein crystal structures to be determined at atomic resolution by using extremely low electron dose. Solving de novel macromolecular structures at atomic resolution has become a routine pipeline for SPA. Its high frame read-out feature enables image processing with dose fractionated movies to correct beam and thermal induced motions, which unprecedently improves image quality. In the last decade, many novel technologies and methodologies have been developed and implemented in SPA, in which the direct electron CMOS detector is the major key breakthrough invention. Intensity decay in high-resolution diffraction shells in series of exposures were employed for measuring radiation damage and resolution reduction as electron dose accumulates. Because impact from electron beams and thermal induced motion significantly exceed radiation damage for cryo-EM single particle analysis (SPA) in the early days, electron crystallography was the primary method to obtain high resolution structure information from 2D membrane and thin 3D protein crystals. When cryo-EM emerged as a powerful tool in structural biology over four decades ago, electron radiation damage to protein crystals was first identified as a major resolution limit factor for high resolution structure determination. More in-depth systematically radiation damage assessments using SPA and MicroED will benefit all applications of cryo-EM, especially cellular structure analysis by tomography. High resolution electron diffraction in MicroED dataset is very sensitive to global damage to 3D protein crystals with low dose accumulation, and its intensity attenuation rates at atomic resolution shell could be applied for estimating ratio of damaged and total selected single particles for SPA. Major differences in data collection and processing scheme are the key factors to different levels of sensitivity to radiation damage at high resolution between the two methods. As a result, the same electron dose could induce much more site-specific damage to macromolecules in 3D protein crystal than single particle samples. Probability of a high energy electron being inelastically scattered by a macromolecule is proportional to number of layers of the molecules in its transmission path. We compared and analyzed these two approaches side-by-side in detail from several aspects to identify some crucial determinants and to explain this discrepancy. Although radiation induced sidechain density loss and resolution decay were observed by both methods, the minimum dose of electron irradiation reducing high-resolution limit reported by SPA is more than ten folds higher than measured by MicroED using the conventional dose concept, and there is a gap between the attained resolutions assessed by these two methods. Single particle analysis (SPA) and micro-crystal electron diffraction (MicroED) have been employed to assess radiation damage with a variety of protein complexes. 2Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, United StatesĮlectron radiation damage to macromolecules is an inevitable resolution limit factor in all major structural determination applications using cryo-electron microscopy (cryo-EM).1Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, United States. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |