Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution

Abstract

Electron cryo-microscopy (cryo-EM) is becoming one of the routine tools for structure determination of biological macromolecules. Commonly, molecular images are obtained by conventional transmission electron microcopy (CTEM) using underfocus and subsequently computationally combined into a high-resolution 3D structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to the cryo-EM test specimen of tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination at 3.5 Angstrom resolution using single-particle based helical reconstruction. A series of cryo-EM TMV maps was resolved at near-atomic resolution taken at different convergence semi-angle (CSA) beams and share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. The associated map B-factors from iDPC-STEM match those obtained by CTEM recordings using 2nd generation direct electron detection devices. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.

Investigating Direct Focused Probe Ptychography for Single Particle Analysis

“…Although recent technological advances have pushed the best resolution achieved in SPA to 1.2 Å [1,2], resolutions around 3 Å are the norm for SPA analysis of proteins. Interestingly this is also around the resolution achieved using SPA with a scanning TEM (STEM) technique known as integrated differential phase contrast (iDPC) [3], closely related to the integrated center of mass (iCoM) signal. Both iDPC and iCoM have recently been used to great effect with various beam sensitive materials.…”

Cryomicroscopy in situ: what is the smallest molecule that can be directly identified without labels in a cell?

“…iDPC-STEM has so far shown the most promise, with a 3.5 Å structure of tobacco mosaic virus having been recently determined using this approach. 101…”

Expanding the cryogenic electron microscopy toolbox to reveal diverse classes of battery solid electrolyte interphase

“…In the former case a real space image of the SEI and lithium can be determined, although this is very difficult to do at atomic resolution, in particular in dose-sensitive materials. 54 4D-STEM techniques applied at different angles is analogous to 3D electron diffraction (or microED) sampling and is potentially very powerful if the sample is sufficiently crystalline.…”