2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from a compact 200 keV cryo-microscope.

Christoph Parthier,Christian Tüting,Fotis L. Kyrilis,Koen M. Visscher,Carmen San Martin,Dmitry A. Semchonok,Ioannis Skalidis,Farzad Hamdi,Lisa Schmidt,Milton T. Stubbs,Annette Meister,Panagiotis L. Kastritis

doi:10.1371/journal.pone.0232540

Christoph Parthier, Christian Tüting + Show 10 more

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https://doi.org/10.1371/journal.pone.0232540

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Abstract

Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®. This is a compact, two-lens illumination system with a constant power objective lens, without any energy filters or aberration correctors, often thought of as a “screening cryo-microscope”. Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We used a quasi-crystallographic reciprocal space approach to fit model coordinates to the experimental cryo-EM map. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (X-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving high resolution. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging can be used to determine structures approaching atomic resolution.

Highlights

Single-particle cryogenic electron microscopy has revolutionized high-resolution structure determination of biomolecular assemblies [1]
The vast majority (94%) of protein complexes solved by cryogenic electron microscopy (cryo-EM) since 2014 are at a resolution lower than 3 Å (Fig 1A); as of July 2019, only 275 high-resolution cryo-EM reconstructions have been deposited in the Electron Microscopy Data Bank (EMDB, https:// www.ebi.ac.uk/pdbe/emdb/), for which 194 atomic models (70.5%) are available in the Protein Data Bank (PDB, https://www.rcsb.org/)
We show that basic settings for automated cryo-electron microscopy imaging using the Glacios1 electron microscope, often thought of as a “screening microscope”, allow structure determination at high resolution

Summary

Introduction

Single-particle cryogenic electron microscopy (cryo-EM) has revolutionized high-resolution structure determination of biomolecular assemblies [1]. The first high-resolution structure better than 3 Å was resolved in 2014 (EMD-6224) and communicated the following year [2]. The vast majority (94%) of protein complexes solved by cryo-EM since 2014 are at a resolution lower than 3 Å (Fig 1A); as of July 2019, only 275 high-resolution (better than 3.0 Å) cryo-EM reconstructions have been deposited in the Electron Microscopy Data Bank (EMDB, https:// www.ebi.ac.uk/pdbe/emdb/), for which 194 atomic models (70.5%) are available in the Protein Data Bank (PDB, https://www.rcsb.org/). Cryo-EM structure of apoferritin at 2.7 Å with the Glacios cryo-microscope. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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