Establishing a multi-scale imaging pipeline on single human cells by correlative cryo-SXT and high-resolution cryo-ET towards understanding of translation regulation

Translation is the process of protein synthesis conducted by the ribosome. Ribosomes distributed across the subcellular space exhibit compositionally and functionally heterogeneity, and in association with ribosome-associated proteins, regulate protein production under varying cellular states. Translation regulation is thus critical to cell homeostasis, and when perturbed, can activate stress response pathways that determine cell survival or death. Understanding how cells respond to stressors at multiple scales, from the level of subcellular organelles (including the ER) to the level of conformational states of the translational machinery, is required and demands the development of multi-scale, label-free, correlative imaging pipelines for the characterization of pristinely-preserved specimens.

Here we aim to combine our strong in house expertise in advanced cryogenic electron tomography (cryo-ET) to visualize large macromolecular complexes inside intact cells at near-atomic resolution, with cryogenic soft X-ray tomography (cryo-SXT) for imaging of subcellular volumes at nanometer-scale resolution. While cryo-ET and cryo-SXT can be performed on human cells vitrified on the same cryo-ET grids, it remains unclear whether the dose spent for generation of high signal-to-noise cryo-SXT data is compatible with high-resolution structural analysis by downstream cryo-ET. In this project, we aim to I) systematically characterize and optimize cryo-SXT imaging conditions which minimally deteriorate the resolution in downstream cryo-ET structural analysis of ribosomes in vitrified cells and II) analyze and correlate the volumetric cryo-SXT and cryo-ET captured on native and translationally-perturbed cells. Cryo-SXT experiments at the MISTRAL beamline at ALBA can be conducted on cells frozen on cryo-ET compatible grids, thereby enabling subsequent correlative cryo-ET on the same specimens and for the first systematic characterization of the effect of cryo-SXT beam damage on the attainable resolution in downstream structural analysis by cryo-ET, leading to the establishment of a multi-scale correlative cryogenic imaging pipeline for exploration of fundamental molecular pathways in health and disease states.