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, Tomasz Uchański, Lingbo Yu, Dimple Karia, Evgeniya V. Pechnikova, Erwin Jong, Jeroen Keizer, Maarten Bischoff, Jamie McCormack, Peter Tiemeijer, Steven W. Hardwick, Dimitri Y. Chirgadze, Garib Murshudov, A. Radu Aricescu () and Sjors H. W. Scheres ()
Additional contact information
Takanori Nakane: MRC Laboratory of Molecular Biology
Abhay Kotecha: Thermo Fisher Scientific
Andrija Sente: MRC Laboratory of Molecular Biology
Greg McMullan: MRC Laboratory of Molecular Biology
Simonas Masiulis: MRC Laboratory of Molecular Biology
Patricia M. G. E. Brown: MRC Laboratory of Molecular Biology
Ioana T. Grigoras: MRC Laboratory of Molecular Biology
Lina Malinauskaite: MRC Laboratory of Molecular Biology
Tomas Malinauskas: University of Oxford
Jonas Miehling: MRC Laboratory of Molecular Biology
Tomasz Uchański: Vrije Universiteit Brussel
Lingbo Yu: Thermo Fisher Scientific
Dimple Karia: Thermo Fisher Scientific
Evgeniya V. Pechnikova: Thermo Fisher Scientific
Erwin Jong: Thermo Fisher Scientific
Jeroen Keizer: Thermo Fisher Scientific
Maarten Bischoff: Thermo Fisher Scientific
Jamie McCormack: Thermo Fisher Scientific
Peter Tiemeijer: Thermo Fisher Scientific
Steven W. Hardwick: University of Cambridge
Dimitri Y. Chirgadze: University of Cambridge
Garib Murshudov: MRC Laboratory of Molecular Biology
A. Radu Aricescu: MRC Laboratory of Molecular Biology
Sjors H. W. Scheres: MRC Laboratory of Molecular Biology

Nature, 2020, vol. 587, issue 7832, 152-156

Abstract: Abstract The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that 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 visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined 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.

Date: 2020
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DOI: 10.1038/s41586-020-2829-0

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