Below 3\xa0\xc5 structure of apoferritin using a multipurpose TEM with a side entry cryoholder

Yoko Kayama,Naoya Terahara,Takayuki Kato,Kazuyoshi Murata,Chihong Song,Raymond N Burton-Smith

doi:10.1038/s41598-021-87183-1

Abstract

Recently, the structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method. Many results of cryo-EM SPA are based on data acquired on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment, although this comes with performance limitations. Here, a multi-purpose TEM with a side entry cryo-holder was used to evaluate the potential of high-resolution SPA, resulting in a 3 Å resolution map of apoferritin with local resolution extending to 2.6 Å. This map clearly showed two positions of an aromatic side chain. Further, examination of optimal imaging conditions depending on two different multi-purpose electron microscope and camera combinations was carried out, demonstrating that higher magnifications are not always necessary or desirable. Since automation is effectively a requirement for large-scale data collection, and augmenting the multi-purpose equipment is possible, we expanded testing by acquiring data with SerialEM using a β-galactosidase test sample. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.

Highlights

Cryo-electron microscopy single particle analysis (SPA) is a technique for reconstructing the threedimensional structure of a biomacromolecule using projected images acquired with an electron m icroscope[1] and was the subject of the Nobel Prize for Chemistry in 2 0172
Focusing on the performance of the electron microscope, including electron source, the sample stage, and the detector is arguably the primary limiting factor. Autoloader stages such as those used in Titan Krios (Thermo Fisher Scientific) and CRYOARM (JEOL) microscopes demonstrate that multiple sample grids can be stored stably for a long period of time, the sample grid can be automatically transported, and data can be automatically collected without manual intervention
We examined the utility of automated acquisition on Setting A. β-galactosidase was used for the sample studied with automation in multipurpose TEM to demonstrate that usable resolutions (Fig. 8A,B) may be achieved with lower symmetry samples compared to higher symmetry apoferritin, which requires more data to achieve similar resolutions

Summary

Introduction

Cryo-electron microscopy (cryo-EM) single particle analysis (SPA) is a technique for reconstructing the threedimensional structure of a biomacromolecule using projected images acquired with an electron m icroscope[1] and was the subject of the Nobel Prize for Chemistry in 2 0172. Autoloader stages such as those used in Titan Krios (Thermo Fisher Scientific) and CRYOARM (JEOL) microscopes demonstrate that multiple sample grids can be stored stably for a long period of time, the sample grid can be automatically transported, and data can be automatically collected without manual intervention. Such a sample stage is difficult to introduce later into a multi-purpose electron microscope and is currently only available pre-installed.

Methods

Results

Conclusion