High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly

Abstract

Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible. To illustrate the electron cryo-tomography workflow for visualizing protein complexes in situ, we describe our workflow of preparing samples, imaging, and image processing using Leginon for data collection, IMOD for image reconstruction, and PEET for subtomogram averaging.

Advances in methods for atomic resolution macromolecular structure determination

“…Cryo-electron tomography (cryo-ET) offers new promise, along with unique challenges of its own. Samples, often of intact cells or tissues, are tilted under low-dose exposure, allowing the reconstruction of three-dimensional images of subcellular structures and their constituent macromolecular assemblies (reviewed in 70 , 71 ). This has led to impressive views of macromolecular machines and the cellular milieu.…”

Techniques and Strategies for Potential Protein Target Discovery and Active Pharmaceutical Molecule Screening in a Pandemic

“…Then, sample images are recorded by microscope with a detector and lens system. Second, after processing the image signal, the three-dimensional structure is obtained by using the three-dimensional reconstruction technology . Compared with other methods, the advantages of cryo-EM technology are not having to prepare crystals and keeping the activation of samples, but it has higher requirements for sample preparation and could not analyze the sample’s components.…”