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Single-particle cryo-EM at atomic resolution

Takanori Nakane, Abhay Kotecha, Andrija Sente, Greg McMullan, Simonas Masiulis, Patricia M.G.E. Brown, Ioana T. Grigoras, Lina Malinauskaite, Tomas Malinauskas, Jonas Miehling, Lingbo Yu, Dimple Karia, Evgeniya V. Pechnikova, Erwin de Jong, Jeroen Keizer, Maarten Bischoff, Jamie McCormack, Peter Tiemeijer, Steven W. Hardwick, Dimitri Y. Chirgadze, Garib Murshudov, A. Radu Aricescu, View ORCID ProfileSjors H.W. Scheres
doi: https://doi.org/10.1101/2020.05.22.110189
Takanori Nakane
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
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Abhay Kotecha
2Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, Eindhoven, 5651 GG, Netherlands
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Andrija Sente
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
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Greg McMullan
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
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Simonas Masiulis
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
5Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, Eindhoven, 5651 GG, Netherlands
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Patricia M.G.E. Brown
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
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Ioana T. Grigoras
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
6Department of Physics, Imperial College London, Prince Consort Road, London, SW7 2BW, UK
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Lina Malinauskaite
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
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Tomas Malinauskas
3Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK
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Jonas Miehling
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
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Lingbo Yu
2Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, Eindhoven, 5651 GG, Netherlands
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Dimple Karia
2Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, Eindhoven, 5651 GG, Netherlands
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Evgeniya V. Pechnikova
2Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, Eindhoven, 5651 GG, Netherlands
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Erwin de Jong
2Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, Eindhoven, 5651 GG, Netherlands
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Jeroen Keizer
2Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, Eindhoven, 5651 GG, Netherlands
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Maarten Bischoff
2Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, Eindhoven, 5651 GG, Netherlands
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Jamie McCormack
2Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, Eindhoven, 5651 GG, Netherlands
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Peter Tiemeijer
2Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, Eindhoven, 5651 GG, Netherlands
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Steven W. Hardwick
4Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA, UK
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Dimitri Y. Chirgadze
4Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA, UK
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Garib Murshudov
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
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A. Radu Aricescu
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
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  • For correspondence: radu@mrc-lmb.cam.ac.uk scheres@mrc-lmb.cam.ac.uk
Sjors H.W. Scheres
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
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  • ORCID record for Sjors H.W. Scheres
  • For correspondence: radu@mrc-lmb.cam.ac.uk scheres@mrc-lmb.cam.ac.uk
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Abstract

The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 Å resolution cryo-EM reconstruction for a prototypical human membrane protein, the β3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 Å resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

Competing Interest Statement

A.K., S.M., L.Y., D.K., E.V.P., E.d.J., J.K., M.B., J.M., and P.T are employees of Thermo Fisher Scientific.

Footnotes

  • ftp://ftp.mrc-lmb.cam.ac.uk/pub/scheres/atomic/

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Single-particle cryo-EM at atomic resolution
Takanori Nakane, Abhay Kotecha, Andrija Sente, Greg McMullan, Simonas Masiulis, Patricia M.G.E. Brown, Ioana T. Grigoras, Lina Malinauskaite, Tomas Malinauskas, Jonas Miehling, Lingbo Yu, Dimple Karia, Evgeniya V. Pechnikova, Erwin de Jong, Jeroen Keizer, Maarten Bischoff, Jamie McCormack, Peter Tiemeijer, Steven W. Hardwick, Dimitri Y. Chirgadze, Garib Murshudov, A. Radu Aricescu, Sjors H.W. Scheres
bioRxiv 2020.05.22.110189; doi: https://doi.org/10.1101/2020.05.22.110189
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Single-particle cryo-EM at atomic resolution
Takanori Nakane, Abhay Kotecha, Andrija Sente, Greg McMullan, Simonas Masiulis, Patricia M.G.E. Brown, Ioana T. Grigoras, Lina Malinauskaite, Tomas Malinauskas, Jonas Miehling, Lingbo Yu, Dimple Karia, Evgeniya V. Pechnikova, Erwin de Jong, Jeroen Keizer, Maarten Bischoff, Jamie McCormack, Peter Tiemeijer, Steven W. Hardwick, Dimitri Y. Chirgadze, Garib Murshudov, A. Radu Aricescu, Sjors H.W. Scheres
bioRxiv 2020.05.22.110189; doi: https://doi.org/10.1101/2020.05.22.110189

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