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

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

doi:10.1371/journal.pone.0232540

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

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

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

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.

Original language	English
Article number	e0232540
Pages (from-to)	1-18
Number of pages	18
Journal	PLoS ONE
Volume	15
Issue number	5
DOIs	https://doi.org/10.1371/journal.pone.0232540
Publication status	Published - 6 May 2020

Funding

This work was supported by the Federal Ministry for Education and Research (BMBF, ZIK program) [grant number 02Z22HN23 to P.L.K.]; the European Regional Development Funds for Saxony-Anhalt [grant number EFRE: ZS/2016/04/ 78115 to P.L.K. and M.T.S.] and the Martin Luther University Halle-Wittenberg. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank Dr. Christos Savva for the excellent apoferritin sample and acknowledge the Thermo Fisher engineers Hartmut Lepuschitz, Christian Heiden, Volker Irmer and Dr. Sonja Welsch for installation of the cryo-microscope and expert technical assistance. The authors also thank engineer Mr. Mohammad Daraei for room preparations and field cancellation installation, Mr. Jürgen Simon and Mr. Ingo Schneider of the MLU as well as Prof. Dr. Reinhardt Paschke, Mr. Matthias Harm and Mr. Grossman of the Biozentrum for the necessary room modifications as well as supervision of the microscope installation, and our IT department, in particular Mr. Patrice Peterson, for excellent maintenance of the IANVS cluster. Funding:ThisworkwassupportedbytheFederal MinistryforEducationandResearch(BMBF,ZIK program)[grantnumber02Z22HN23toP.L.K.]; theEuropeanRegionalDevelopmentFundsfor Saxony-Anhalt[grantnumberEFRE:ZS/2016/04/ 78115toP.L.K.andM.T.S.]andtheMartinLuther

Funders	Funder number
European Regional Development Funds for Saxony-Anhalt	ZS/2016/04/ 78115
Martin Luther University Halle-Wittenberg
MinistryforEducationandResearch
Saxony-Anhalt	ZS/2016/04/ 78115toP
Thermo Fisher engineers Hartmut Lepuschitz
Bundesministerium für Bildung und Forschung	02Z22HN23

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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@article{6be03bee3ecc4a2c9e0799bd1065215f,

title = "2.7 {\AA} cryo-EM structure of vitrified M. Musculus H-chain apoferritin from a compact 200 keV cryo-microscope",

abstract = "Here we present the structure of mouse H-chain apoferritin at 2.7 {\AA} (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios{\textregistered}. 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.",

author = "Farzad Hamdi and Christian T{\"u}ting and Semchonok, {Dmitry A.} and Visscher, {Koen M.} and Kyrilis, {Fotis L.} and Annette Meister and Ioannis Skalidis and Lisa Schmidt and Christoph Parthier and Stubbs, {Milton T.} and Kastritis, {Panagiotis L.}",

year = "2020",

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AU - Hamdi, Farzad

AU - Tüting, Christian

AU - Semchonok, Dmitry A.

AU - Visscher, Koen M.

AU - Kyrilis, Fotis L.

AU - Meister, Annette

AU - Skalidis, Ioannis

AU - Schmidt, Lisa

AU - Parthier, Christoph

AU - Stubbs, Milton T.

AU - Kastritis, Panagiotis L.

PY - 2020/5/6

Y1 - 2020/5/6

N2 - 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.

AB - 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.

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

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