Cryo-electron microscopy (cryo-EM) helps reveal the elusive mechanism of transmembrane proton transport. Cryo-EM determined the structure of a yeast vacuole Vo proton channel pump complex at 2.7 Å resolution (15 polypeptides shown in different colors), specific protein-lipid interactions, and the role of water in the dynamic process. [PDB ID: 6MOR. Roh, S. H., et al. 2020. "Cryo-EM and MD infer water-mediated proton transport and autoinhibition mechanisms of Vo complex," Science Advances 6(41), eabb9605. DOI: 10.1126/sciadv.abb9605.]
Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) techniques use electrons to provide images of biological materials frozen in their native state. Materials range from proteins and nucleic acids to very large biological assemblies and complexes, and image resolutions range from nanometer to atomic scales.
Collectively a suite of sample preparation techniques, 200-300 kV electron microscopes, and data analysis tools is available at DOE user facilities to address a range of imaging needs for BER-mission-relevant biological research. Cryo-EM uniquely captures dynamic ensembles of macromolecular structures as they occur in solution. Image analysis can then separate these ensembles into high-resolution snapshots that capture their compositional and conformational variance and their dynamics.
Cryo-ET is an emerging technique that can resolve subcellular structures inside cells and tissues. It can achieve nanometer-scale resolution for the entire sample and atomic-scale resolution for abundant molecular components in situ when post-tomographic data processing is added. Cryo-fluorescence light microscopy (cryo-FLM) and subsequent cryo-ET of frozen, hydrated cells can be used to label specific proteins and study cellular and molecular functions and dynamics in the 3D context of cells and tissues at a higher resolution than any other imaging techniques. Cryo-ET can also be preceded by cryogenic focused ion beam scanning electron microscopy (cryo-FIB-SEM), to produce lamellae from vitrified cells that are thin enough for cryo-ET imaging.
Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. A pipeline integrating three imaging modalities revealed the distribution of subcellular structures in yeast cells: cryo-fluorescence confocal microscopy, volume cryogenic focused ion beam scanning electron microscopy (volume cryo-FIB-SEM), and transmission cryo-ET. Cell membrane and wall (yellow) enclose the nucleus (orange), mitochondria (purple), vacuoles (red), and other subcellular structures. [Reprinted with permission from Elsevier from Wu, G-H., et al. 2020. "Multi-scale 3D Cryo-Correlative Microscopy for Vitrified Cells," Structure 28(11) 1232–37. DOI: 10.1016/j.str.2020.07.017. Copyright 2020.]
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