May, M. B. | Search bioRxiv

bioRxiv2025

Capturing ribosomal structures in cellular extracts with cryoPRISM: A purification-free cryoEM approach reveals novel structural states

Structural analyses of ribosomes by single particle cryogenic electron microscopy (cryoEM) have traditionally relied on purified or reconstituted samples, with particles often trapped in desired states using genetic, pharmacological, or biochemical perturbations. While informative, such in vitro methods often fail to capture the full diversity of structural states and associated protein factors present in cells. In contrast, in situ cryoelectron tomography preserves cellular context but is limited by low throughput and modest resolution. Here, we present cryoPRISM (purification-free ribosome imaging from subcellular mixtures), a rapid ex vivo workflow encompassing cell lysis, vitrification, and image analysis methods for high-resolution analyses of ribosomal structures directly from cell lysates. Applying cryoPRISM in E. coli, we resolved more than twenty distinct ribosomal states spanning assembly, translation initiation, elongation, trans-translation, and quiescence, including a novel configuration of EF-G bound to idle ribosomes with the ribosome hibernation factor RaiA. Given its speed, accessibility, and ability to preserve native interactions and structural heterogeneity, we anticipate that cryoPRISM will be broadly applicable for uncovering ribosomal biology across diverse organisms and conditions.

biochemistry↗

bioRxiv2023

Application of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination

In cryogenic electron microscopy (cryo-EM), purified macromolecules are typically applied to a grid bearing a holey carbon foil, blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice that is suspended across roughly 1 m-wide foil holes. The resulting sample is then imaged using cryogenic transmission electron microscopy and, after substantial image processing, near-atomic resolution structures can be determined. Despite cryo-EMs widespread adoption, sample preparation remains a severe bottleneck in cryo-EM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryo-EM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryo-EM grids, and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 [A] resolution density map of an exemplar Cas9 complex using a highly pure sample at a relatively low concentration. SUMMARYThe application of support layers, such as graphene, to cryo-electron microscopy grids can increase the density of particles imaged, limit particle interactions with the air-water interface, reduce the extent of beam-induced motion, and, in some instances, improve the distribution of particle orientations. This paper describes a robust protocol for coating cryo-EM grids with a monolayer of graphene for improved cryo-sample preparation.

biochemistry↗