Achieving Near-Atomic Resolution in Single-Particle Cryo-EM Using a Single Micrograph

Abstract

Single-particle cryo-electron microscopy (cryo-EM) typically requires the acquisition and analysis of a large number of micrographs to achieve high-resolution three-dimensional (3D) reconstructions of biological macromolecules. In this study, we demonstrate that when sample quality is sufficiently high, near-atomic resolution 3D density maps can be obtained using particle images from a single cryo-EM micrograph. Using this approach, we successfully reconstructed the 3D structures of apoferritin and the 20S proteasome from single-micrograph datasets. Our analysis reveals that the zero-crossings in the single-micrograph contrast transfer function (CTF) can be compensated by variations in the axial (Z-axis) distribution of particles within the vitrified sample and the intrinsic astigmatism of the optical system, thereby effectively recovering structural information across the full range of spatial frequencies. Furthermore, by analyzing reconstructions from dose-fractionated frames with varying cumulative electron exposures, we found that the minimal electron dose required to preserve undamaged high-frequency information while maintain sufficient low-frequency signals for accurate orientation determination is substantially lower than the commonly applied total doses. Based on this, we estimate the theoretical lower bounds of both electron dose and particle number required for high-resolution structure determination. This work advances our understanding of signal preservation in cryo-EM imaging and provides practical insight for optimizing data acquisition strategies to maximize retention of high-frequency structural information.