Advances in Single-Particle Electron Cryomicroscopy Structure Determination applied to Sub-tomogram Averaging

TLDR

Recent advances in specimen preparation, data collection, and image processing have enhanced structure determination by single‑particle cryo‑EM. The study investigates how these advances can be applied to improve structures obtained by cryo‑ET and sub‑tomogram averaging. A new 3‑D contrast‑transfer‑function model was incorporated into RELION’s regularized likelihood optimization, and single‑particle analysis combined with sub‑tomogram averaging of direct‑electron‑detector data was used to assess radiation‑induced specimen movements. The authors observed substantial sample movements during tomographic acquisition that were markedly reduced on ultrastable gold substrates, enabling a sub‑nanometer resolution structure of the hepatitis B capsid and highlighting the importance of minimizing radiation‑induced motion for higher tomogram quality.

Abstract

Recent innovations in specimen preparation, data collection, and image processing have led to improved structure determination using single-particle electron cryomicroscopy (cryo-EM). Here we explore some of these advances to improve structures determined using electron cryotomography (cryo-ET) and sub-tomogram averaging. We implement a new three-dimensional model for the contrast transfer function, and use this in a regularized likelihood optimization algorithm as implemented in the RELION program. Using direct electron detector data, we apply both single-particle analysis and sub-tomogram averaging to analyze radiation-induced movements of the specimen. As in single-particle cryo-EM, we find that significant sample movements occur during tomographic data acquisition, and that these movements are substantially reduced through the use of ultrastable gold substrates. We obtain a sub-nanometer resolution structure of the hepatitis B capsid, and show that reducing radiation-induced specimen movement may be central to attempts at further improving tomogram quality and resolution.