New Cryo-EM Polishing Technique Enables Near-Atomic Resolution Imaging of Proteins in Living Cells
Researchers developed a cryogenic low-energy polishing method for focused ion beam microscopy that significantly reduces surface damage to cellular samples, enabling near-atomic resolution imaging of proteins as small as 400 kDa directly inside cells. Previously, in situ cryo-electron microscopy at near-atomic resolution was largely limited to very large complexes like ribosomes, because the ion beam used to thin cell samples caused subsurface damage that degraded data quality. The advance could allow scientists to study how protein structures change in response to different cellular conditions, opening new possibilities for structural biology and drug discovery.
A team of researchers has developed a cryogenic low-energy polishing technique for cryo-focused ion beam (cryo-FIB) microscopy that reliably produces thin cellular lamellae with minimal subsurface damage across different cell types. This damage had previously been a major bottleneck preventing near-atomic resolution imaging of smaller proteins in their native cellular environment, restricting such analyses largely to exceptionally large complexes like ribosomes. By combining the new polishing approach with in situ single-particle analysis, the researchers pushed the molecular weight lower limit for near-atomic in situ reconstruction down to approximately 400 kDa. They demonstrated the method by resolving several protein complexes in the green alga Chlamydomonas reinhardtii, including photosynthetic complexes at 3.3–3.4 Ångström resolution, metabolic enzymes at 3.3 Å, a chloroplast ribosome at 4.0 Å, and respiratory chain complexes at 3.7 Å. The workflow is also efficient enough to enable rapid structural comparisons across different cellular states, making multi-condition in situ structural studies more practical.
What's missing
As a preprint posted on bioRxiv, this work has not yet undergone formal peer review, so the methods and conclusions have not been independently validated by the scientific community. The authors do not discuss the lower bound of molecular weight that might ultimately be achievable, nor the computational or instrument costs associated with scaling the workflow.
What different sources said
- bioRxivCenter
Low-Energy Polishing Facilitates Breaking the Resolution Barrier of In Situ Cryo-EM
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