Coma-corrected rapid single-particle cryo-EM data collection on the CRYO ARM 300.

Annelore Stroobants,Rouslan G Efremov

doi:10.1107/s2059798321002151

Abstract

Single-particle cryogenic electron microscopy has recently become a major method for determining the structures of proteins and protein complexes. This has markedly increased the demand for throughput of high-resolution electron microscopes, which are required to produce high-resolution images at high rates. An increase in data-collection throughput can be achieved by using large beam-image shifts combined with off-axis coma correction, enabling the acquisition ofmultiple images from a large area of the EM grid without moving the microscope stage. Here, the optical properties of the JEOL CRYO ARM 300 electron microscope equipped with a K3 camera were characterized under off-axis illumination conditions. It is shown that efficient coma correction can be achieved for beam-image shifts with an amplitude of at least 10 µm, enabling a routine throughput for data collection of between 6000 and 9000 images per day. Use of the benchmark for the rapid data-collection procedure (with beam-image shifts of up to 7 µm) on apoferritin resulted in a reconstruction at a resolution of 1.7 Å. This demonstrates that the rapid automated acquisition of high-resolution micrographs is possible using a CRYO ARM 300.

Highlights

Density maps of proteins can be reconstructed at atomic resolution using single-particle cryogenic electron microscopy
Ðtcycle=nÞ þ timg where n is the number of micrographs recorded per stage position, tcycle = tstage + tfocus + tbeam + tice is the cumulative time of operations performed only once per stage position, and timg = tBS + tstab + trec + tSerialEM is the cumulative time for collecting one micrograph
We characterized the performance of the CRYO ARM 300 electron microscope for single-particle data collection using coma- and astigmatism-corrected image shifts with amplitudes of up to 7 mm

Summary

Introduction

Density maps of proteins can be reconstructed at atomic resolution using single-particle cryogenic electron microscopy (cryo-EM; Nakane et al, 2020; Yip et al, 2020). Intense development of the method continues, single-particle cryo-EM has already revolutionized structural biology over the course of the last six years. High-resolution imaging imposes stringent constraints on electron microscopes, as images with minimal subatomic drift and minimal aberrations are required to preserve information at atomic resolution ($1 A ). The development of dedicated high-resolution, stable and fully automated electron microscopes, which are equipped with energy filters and produce highly coherent parallel illumination over large illumination areas, has played a critical role in the success of the method

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Conclusion