Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV

Mengyu Wu,Gabriel C Lander,Mark A Herzik

doi:10.1016/j.yjsbx.2020.100020

Abstract

Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination of well-behaved specimens to high-resolution, there nonetheless remains a discrepancy between the resolutions attained for biological specimens and the information limits of modern transmission electron microscopes (TEMs). Instruments operating at 300 kV equipped with DEDs are the current paradigm for high-resolution single-particle cryo-EM, while 200 kV TEMs remain comparatively underutilized for purposes beyond sample screening. Here, we expand upon our prior work and demonstrate that one such 200 kV microscope, the Talos Arctica, equipped with a K2 DED is capable of determining structures of macromolecules to as high as ∼1.7 Å resolution. At this resolution, ordered water molecules are readily assigned and holes in aromatic residues can be clearly distinguished in the reconstructions. This work emphasizes the utility of 200 kV electrons for high-resolution single-particle cryo-EM and applications such as structure-based drug design.

Highlights

High-resolution three-dimensional (3D) structure determination of vitrified biological macromolecules by cryogenic electron microscopy single-particle analysis (SPA) has expanded the utility of the structural biology field, enabling detailed visualization of targets that were previously intractable using other structural techniques (Bai et al, 2015; Dambacher et al, 2016; de la Pena et al, 2018; Zhan et al, 2018)
We previously determined the ∼2.6 Å resolution structure of rabbit muscle aldolase using a base-model Thermo Fisher Scientific Talos Arctica equipped with a Gatan K2 Summit direct electron detectors (DEDs), demonstrating that high-resolution reconstructions are attainable using a two-condenser lens transmission electron microscopes (TEMs) operating at 200 kV (Herzik et al, 2017b)
Despite the ubiquity of single particle cryogenic electron microscopy (cryo-EM), there is a lingering misconception that high-resolution structure determination of biological targets can only be achieved with 300 kV three-condenser lens instruments such as the Titan Krios, with 200 kV instrumentation primarily relegated to sample screening

Summary

Introduction

High-resolution three-dimensional (3D) structure determination of vitrified biological macromolecules by cryogenic electron microscopy (cryo-EM) single-particle analysis (SPA) has expanded the utility of the structural biology field, enabling detailed visualization of targets that were previously intractable using other structural techniques (Bai et al, 2015; Dambacher et al, 2016; de la Pena et al, 2018; Zhan et al, 2018). Due to significant advancements in instrumentation, data collection software, and data processing algorithms, obtaining reconstructions of sufficiently high resolution for reliable model building (i.e., 3 Å resolution or better) has become increasingly routine using SPA, and has garnered substantial interest in the achievable resolution limits of this technique Though this resolution regime represents an impressive feat, there remains a considerable discrepancy between what has been presently achieved for structural studies of biological macromolecules (e.g., adeno-associated virus (Tan et al, 2018), beta-galactosidase (Bartesaghi et al, 2015; Bartesaghi et al, 2018), glutamate dehydrogenase (Merk et al, 2016), apoferritin (Danev et al, 2019; Hamaguchi et al, 2019; Zivanov et al, 2018)) versus theoretically attainable resolutions possible with a transmission electron microscope (TEM) (i.e., the Abbe resolution limit given the wavelength of the electron beam). Highresolution information of the specimen substantially deteriorates during imaging due to accumulation of beam-induced radiation damage

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Results

Conclusion