Single-particle cryo-EM at atomic resolution

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.

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Fig. 1: New imaging technologies for cryo-EM.
Fig. 2: GABAAR reconstructions.
Fig. 3: Apoferritin reconstruction.

Data availability

Atomic coordinates for the GABAAR construct and apoferritin have been deposited in the Protein Data Bank with accession codes 7A5V and 7A4M, and the cryo-EM density maps have been deposited in the Electron Microscopy Data Bank with accession codes EMD-11657 and EMD-11638, respectively. Raw movies of all datasets have been deposited in the Electron Microscopy Public Image Archive (https://www.ebi.ac.uk/pdbe/emdb/empiar/) with accession codes EMPIAR-10500 for GABAAR and EMPIAR-10424 for apoferritin.

Code availability

Both the implementation of reading EER movies into RELION and the EERformat itself have been distributed as free, open-source software, with downloads available from https://github.com/3dem/relion and https://github.com/fei-company/EerReaderLib. The in-house-generated Linux port of the BoxNet2D neural network in Warp is available from https://gist.github.com/biochem-fan/76ff5934495585da56ab2c0af4fe2563.

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Acknowledgements

We thank M. van Beers, M. Veerhoek and R. Jonkers for maintaining the Titan Krios microscopes; W. van Dijk, B. van de Kerkhof, S. Konings and G. van Duinen for advice on optics and microscope alignments; B. van Knippenberg, A. Voigt and F. Grollios for support with EPU software; H. Yanagisawa from the Kikkawa laboratory for the apoferritin sample; P. Miller for the GABAA expression vectors; K. Yamashita for atomic model refinement; A. Koh, T. Darling and J. Grimmett for support with computing; G. Lezcano Singla and E. Franken for support with the EER format; G. van Hoften and G. Hosmar for support with the Falcon-4 camera; and E. Ioannou for logistics support. This work was supported by the EM facilities at the MRC-LMB, the Biochemistry Department of Cambridge University and Thermo Fisher Scientific. The Cryo-EM Facility at Department of Biochemistry is funded by the Wellcome Trust (206171/Z/17/Z; 202905/Z/16/Z) and the University of Cambridge. We acknowledge funding from the UK Medical Research Council (MC_UP_A025_1012 to G. Murshudov, MR/L009609/1 and MC_UP_1201/15 to A.R.A. and MC_UP_A025_1013 to S.H.W.S.); the Japan Society for the Promotion of Science (Overseas Research Fellowship to T.N.); MRC-LMB, Cambridge Trust and School of Clinical Medicine, University of Cambridge (LMB Cambridge Scholarship and Cambridge MB/PhD fellowship to A.S.); the European Commission (Marie Skłodowska-Curie Actions H2020-MSCA-IF-2015/709054 to L.M. and H2020-MSCA-IF-2017/793653 to P.M.G.E.B.); EMBO (long term fellowship 300-2015 to L.M.); Cancer Research UK (T.M., grants C20724/A14414 and C20724/A26752 to C. Siebold, University of Oxford); Boehringer Ingelheim Fonds (PhD Fellowship to J. Miehling; and the Research Foundation – Flanders (FWO, PhD Fellowship to T.U.).

Author information

Affiliations

Authors

Contributions

E.d.J., J.K., M.B., J. McCormack and P.T. contributed to microscope hardware and software developments; G. McMullan performed DQE analysis; D.K., A.S. and S.M. prepared cryo-EM grids; T.U. produced megabodies; A.K., L.Y., E.V.P., S.W.H. and D.Y.C. acquired cryo-EM data; T.N., A.K., A.S., L.Y., S.M., P.M.G.E.B., I.T.G., L.M., J. Miehling and S.H.W.S. analysed cryo-EM data; T.N., A.R.A., T.M. and G. Murshudov prepared atomic models; T.N., A.K., A.R.A. and S.H.W.S. conceived the project and designed the experiments; G.M., A.R.A. and S.H.W.S. wrote the manuscript, with input from all authors.

Corresponding authors

Correspondence to A. Radu Aricescu or Sjors H. W. Scheres.

Ethics declarations

Competing interests

A.K., S.M., L.Y., D.K., E.V.P., E.d.J., J.K., M.B., J. McCormack and P.T. are employees of Thermo Fisher Scientific. The other authors declare no competing interests.

Additional information

Peer review information Nature thanks Serban Ilca, Henning Stahlberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Characteristics of the new cryo-EM technology.

a, Four consecutive measurements of the CFEG beam current over a period of nine hours. The FEG tip was flashed just before the start of each measurement. b, The dose at the sample as measured for the same four experiments in a.

Extended Data Fig. 2 Cryo-EM for GABAAR.

a, B-factor plots for three datasets using an X-FEG, the new energy filter with a slit width of 3eV (orange), 5eV (blue) and 10 eV (grey) and a Falcon-4 camera. b, Two orthogonal views of an electron tomogram for ice thickness measurement. Scale bar is 50 nm. c, Representative electron micrograph from the CFEG dataset. Scale bar is 30 nm. d, A selection of 2D class average images. e, Local resolution map. f, Fourier Shell Correlation (FSC) between the two independently refined half-maps. g, FSC between the model and the map calculated for the model refined against the full reconstruction (black); the model refined in the first half-map against that half-map (FSCwork; blue); and the model refined in the first half-map against the second half-map (FSCtest; dashed orange). Atomic models were refined including spatial frequencies up to 1.63 Å.

Extended Data Fig. 3 GABAAR reconstruction details.

a, The GABAAR cryo-EM map viewed from the extracellular space (top view). The density of one subunit of the homopentamer is highlighted in yellow; glycans are orange; the nanobody domain of Mb25 green; and the lipid nanodisc light blue. b, As in a, but viewed parallel to the plasma membrane space (side view). c, Stacking of histamine with aromatic residues in the ligand-binding pocket. Water molecules removed for clarity. d, Radiation damage in the ligand-binding pocket illustrated by difference maps of Asp43 and Glu155 carboxyl groups. e, Radiation damage causes partial reduction of the disulfide bond between Cys136 and Cys150. Left panel: oxidized state, right panel: reduced state. Only Cα, Cβ and sulfur atoms depicted for clarity. f, Difference map revealing the hydrogen bonding network between β-strands. g, A close-up view of the lipids (blue) surrounding the transmembrane region of the receptor. Compared to a, b, the contour level is decreased. Red arrow indicates section level as depicted in h. The general anaesthetics pocket and the neurosteroid modulation site are indicated by the red circle and rectangle, respectively. h, Top-view of a transverse section through the GABAAR transmembrane region shows semi-ordered lipids surrounding individual subunits. i, Close-up view of the general anaesthetics pocket. j, Close-up view of the neurosteroid modulation site. Lipids in i and j could not be identified unambiguously and therefore are modelled as aliphatic chains.

Extended Data Fig. 4 Cryo-EM for apoferritin.

a, Representative electron micrograph. Scale bar is 20 nm. b, 2D class average images. c, Local resolution map. d, Fourier Shell Correlation (FSC) between the two independently refined half-maps. e, FSC between the model and the map as calculated for the model refined against the full reconstruction against that map (black); the model refined in the first half-map against that half-map (FSCwork; blue); and the model refined in the first half-map against the second half-map (FSCtest; dashed orange). FSCwork and FSCtest curves are shown for three different models: with hydrogens and anisotropic B-factors (solid lines); with hydrogens and isotropic B-factors (dashed lines); and without hydrogens and with isotropic B-factors (dotted lines). Including hydrogens and using anisotropic B-factors gives a better fit to the data at medium resolution, but lead to a small amount of overfitting at high resolution. Atomic models were refined including spatial frequencies up to 1.15 Å. f, The beam tilt in X (blue) and Y (orange); the resolution (res.) for subsets of 5,000 consecutive particles (black); and RELION's rlnGroupScaleCorrection values for individual micrographs (blue dots) during the data acquisition experiment. Events like CFEG flashing (red), liquid nitrogen filling (black) and moving the grid to another grid square (dark blue) are indicated with vertical lines. The cutoff in scale correction values used to discard part of the micrographs is indicated with a dashed grey line.

Extended Data Fig. 5 Electrostatic potential of hydrogen atoms.

a, Calculated profile (see Methods) of the electrostatic scattering potential along the bond between a carbon and a hydrogen atom, for a B-value of 10 Å2 and resolutions (d) of 1.0 Å (orange), 1.2 Å (blue), 1.5 Å (grey) and 1.8 Å (black). The two vertical lines indicate the electron-carbon distance of 0.98 Å and the proton-carbon distance of 1.09 Å. b, As in a, but for a resolution of 1.2 Å and B-values of 5 Å2, 10 Å2, 15 Å2 and 20 Å2. c, Distances from the carbon atom (in Å) at which the calculated profile of the electrostatic scattering potential along the bond between a carbon and a hydrogen atom is at its maximum, for different resolutions (d) and B-values.

Extended Data Table 1 Cryo-EM data collection parameters
Extended Data Table 2 Refinement and validation statistics

Supplementary information

Reporting Summary

Video 1

Dose-dependent evolution of the cryo-EM density in the GABAAR ligand-binding pocket.

Video 2

Dose-dependent evolution of the cryo-EM density of the disulfide bond between cysteines 136 and 150.

Video 3

Overview of the apoferritin cryo-EM density.

Video 4

Dose-dependent evolution of the cryo-EM density in the apoferritin map.

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Nakane, T., Kotecha, A., Sente, A. et al. Single-particle cryo-EM at atomic resolution. Nature 587, 152–156 (2020). https://doi.org/10.1038/s41586-020-2829-0

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