Al-Amoudi, A.; Baradaran, R.; Yuan, X.; Naschberger, A.

In situ high-resolution structure determination is limited to samples that are thin enough to be penetrated by the electron beam during imaging. Currently, the most widely used method for sample thinning involves focused ion or plasma beam milling of biological specimens to produce lamellae with thicknesses as low as 100-150 nm. However, surface damage caused by the milling process can extend 30-60 nm deep, leaving only a narrow undamaged core and thereby restricting the usable lamella thickness. This imposes significant limitations on single-particle analysis of smaller macromolecular complexes due to elevated structural noise, which cannot be avoided in situ because of the dense cellular environment composed of proteins, membranes, and other ultrastructures. Therefore, alternative methods capable of producing thinner samples are needed to reduce this structural background and enable in situ single-particle analysis. Here, we demonstrate that high-resolution structures at side-chain level can be obtained from vitreous sections prepared by cryo-ultramicrotomy, both in vitro and in situ. We optimized the method to produce high-quality sections as thin as 30-50 nm, free from significant surface damage such as crevasses. Using this approach, we determined the structure of the 50S ribosomal subunit in vitro in a standard buffer system and show that orientation bias can be overcome in using this technique. In addition, we systematically analyzed section thickness and show that our ultrathin sections are thin enough to physically cleave 70S ribosomes, suggesting that this approach may be particularly well suited for the structural analysis of smaller macromolecules. Finally, we present a sub-3 A resolution reconstruction of the 50S ribosomal large subunit in situ from bacterial cells. Future directions and potential applications are discussed, with a focus on advancing in situ single-particle analysis of smaller macromolecular complexes.