Pechnikova, E. V. | bioRxiv Science

bioRxiv2021

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

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 [A] 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.

biophysics↗

bioRxiv2020

Single-particle cryo-EM at atomic resolution

The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 [A] resolution cryo-EM reconstruction for a prototypical human membrane protein, the {beta}3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 [A] resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

biophysics↗