Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

Abstract

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo- EM) image processing has been well documented for single particle analysis (SPA). Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Angstrom resolution range. In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Angstrom. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.

CryoVesNet: A Dedicated Framework for Synaptic Vesicle Segmentation in Cryo Electron Tomograms

“…Cellular structures are preserved down to the atomic scale through vitrification and observation of the samples in a fully hydrated state. When a macromolecule is present in a sufficient number of copies in the cells imaged by cryo-ET, it is possible to obtain its atomic structure in situ using subtomogram averaging [2,3]. Cellular cryo-ET datasets are usually extremely complex, making them difficult to analyze.…”