Strategies for studying discrete heterogeneity in situ using cryo-electron tomography Structural variability plays a crucial role in enabling biological function, as the ability of proteins to adopt multiple conformations allows them to perform diverse cellular tasks. Cryo-electron tomography combined with subtomogram averaging and classification has emerged as a powerful technique for elucidating the conformational dynamics of proteins in their…
In situ structure determination of conformationally flexible targets using nextPYP Single-particle cryoelectron tomography (SP-CET) is an imaging technique capable of determining the structure of proteins in their cellular environment at high-resolution. nextPYP is a web-based application designed to streamline the SP-CET structure determination process and facilitate the analysis of conformational variability. Here we explain how to use nextPYP-based methods…
MiLoPYP: self-supervised molecular pattern mining and particle localization in situ Cryo-electron tomography (CET) allows the routine visualization of cellular landscapes in three dimensions at nanometer-range resolutions. When combined with single-particle tomography (SPT), it is possible to obtain near-atomic resolution structures of frequently occurring macromolecules within their native environment. Two outstanding challenges associated with CET/SPT are the automatic identification and…
Advances in cryo-ET data processing: meeting the demands of visual proteomics Cryogenic electron tomography (cryo-ET), a method that enables the viewing of biomolecules in near-native environments at high resolution, is rising in accessibility and applicability. Over the past several years, once slow sample preparation and data collection procedures have seen innovations which enable rapid collection of the large datasets…
Accurate size-based protein localization from cryo-ET tomograms Cryo-electron tomography (cryo-ET) combined with sub-tomogram averaging (STA) allows the determination of protein structures imaged within the native context of the cell at near-atomic resolution. Particle picking is an essential step in the cryo-ET/STA image analysis pipeline that consists in locating the position of proteins within crowded cellular tomograms so that they…
Joint micrograph denoising and protein localization in cryo-electron microscopy Cryo-electron microscopy (cryo-EM) is an imaging technique that allows the visualization of proteins and macromolecular complexes at near-atomic resolution. The low electron doses used to prevent radiation damage to the biological samples result in images where the power of noise is 100 times stronger than that of the signal. Accurate…
nextPYP: a comprehensive and scalable platform for characterizing protein variability in situ using single-particle cryo-ET Single-particle cryo-electron tomography (SP-CET) is an emerging technique capable of determining the structure of proteins imaged within the native context of cells at molecular resolution. While high-throughput techniques for sample preparation and tilt-series acquisition are beginning to provide sufficient data to allow structural studies…
Multiple-image super-resolution of cryo-electron micrographs based on deep internal learning Single-particle cryo-electron microscopy (cryo-EM) is a powerful imaging modality capable of visualizing proteins and macromolecular complexes at near-atomic resolution. The low electron-doses used to prevent radiation damage to the biological samples, however, result in images where the power of the noise is 100 times greater than the power of…
