Abstract
Cryo-electron microscopy (cryo-EM) has become an indispensable tool for structural studies of biological macromolecules. Two additional predominant methods are available for studying the architectures of multiprotein complexes: 1) single-particle analysis of purified samples and 2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, whereas the latter can capture proteins in their native state but has a low signal-to-noise ratio and yields lower-resolution structures. Here, we present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos. Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. Moreover, we investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts.
Highlights
Cryo-electron microscopy has become an indispensable tool for structural studies of biological macromolecules
We present an approach for structural characterization of protein complexes isolated from single cells engaged in development
We demonstrate that a single cell contains a sufficient number of protein particles to enable structural characterization by EM
Summary
We present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. We investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts. We find that the number of ribosomes per polysome remains consistent between early- and late-stage embryos These results demonstrate the potential of EM for structural characterization of unpurified macromolecular machines obtained from samples as small as a single cell
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have