Preparation of oxygen-sensitive proteins for high-resolution cryoEM structure determination using blot-free vitrification.

ABSTRACT: High-quality grid preparation for single-particle cryogenic electron microscopy (cryoEM) remains a bottleneck for routinely obtaining high-resolution structures. The issues that arise from traditional grid preparation workflows are particularly exacerbated for oxygen-sensitive proteins, including metalloproteins, whereby oxygen-induced damage and alteration of oxidation states can result in protein inactivation, denaturation, and/or aggregation. Indeed, 99% of the current structures in the EMBD were prepared aerobically and limited successes for anaerobic cryoEM grid preparation exist. Current practices for anaerobic grid preparation involve a vitrification device located in an anoxic chamber, which presents significant challenges including temperature and humidity control, optimization of

SUBMITTER: Cook BD 

PROVIDER: S-EPMC11997128 | biostudies-literature | 2025 Apr

REPOSITORIES: biostudies-literature