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Achieving better-than-3-Å resolution by single-particle cryo-EM at 200 keV

Nature Methods volume 14, pages 1075–1078 (2017)Cite this article

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Abstract

Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.

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Figure 1: Cryo-EM reconstructions of the 20S proteasome at ∼3.1-Å resolution.
Figure 2: Structure of aldolase at ∼2.6-Å resolution.

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References

  1. Merk, A. et al. Cell 165, 1698–1707 (2016).

    Article  CAS  Google Scholar 

  2. Danev, R., Tegunov, D. & Baumeister, W. eLife 6, e23006 (2017).

    Article  Google Scholar 

  3. Henderson, R. Q. Rev. Biophys. 28, 171–193 (1995).

    Article  CAS  Google Scholar 

  4. Campbell, M.G. et al. J. Struct. Biol. 188, 183–187 (2014).

    Article  CAS  Google Scholar 

  5. Li, X. et al. J. Mol. Biol. 429, 79–87 (2017).

    Article  CAS  Google Scholar 

  6. Glaeser, R.M., Typke, D., Tiemeijer, P.C., Pulokas, J. & Cheng, A. J. Struct. Biol. 174, 1–10 (2011).

    Article  Google Scholar 

  7. Suloway, C. et al. J. Struct. Biol. 151, 41–60 (2005).

    Article  CAS  Google Scholar 

  8. Lander, G.C. et al. J. Struct. Biol. 166, 95–102 (2009).

    Article  CAS  Google Scholar 

  9. Russo, C.J. & Passmore, L.A. Science 346, 1377–1380 (2014).

    Article  CAS  Google Scholar 

  10. Zheng, S.Q. et al. Nat. Methods 14, 331–332 (2017).

    Article  CAS  Google Scholar 

  11. Rohou, A. & Grigorieff, N. J. Struct. Biol. 192, 216–221 (2015).

    Article  Google Scholar 

  12. Zhang, K. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  Google Scholar 

  13. Kimanius, D., Forsberg, B.O., Scheres, S.H. & Lindahl, E. eLife 5, e18722 (2016).

    Article  Google Scholar 

  14. Henderson, R. et al. Structure 20, 205–214 (2012).

    Article  CAS  Google Scholar 

  15. Campbell, M.G., Veesler, D., Cheng, A., Potter, C.S. & Carragher, B. eLife 4, e06380 (2015).

    Article  Google Scholar 

  16. Egerton, R.F. Ultramicroscopy 145, 85–93 (2014).

    Article  CAS  Google Scholar 

  17. Campbell, M.G. et al. Structure 20, 1823–1828 (2012).

    Article  CAS  Google Scholar 

  18. Li, X. et al. Nat. Methods 10, 584–590 (2013).

    Article  CAS  Google Scholar 

  19. Rubinstein, J.L. & Brubaker, M.A. J. Struct. Biol. 192, 188–195 (2015).

    Article  Google Scholar 

  20. Grant, T. & Grigorieff, N. eLife 4, e06980 (2015).

    Article  Google Scholar 

  21. Scheres, S.H. eLife 3, e03665 (2014).

    Article  Google Scholar 

  22. Cheng, Y., Grigorieff, N., Penczek, P.A. & Walz, T. Cell 161, 438–449 (2015).

    Article  CAS  Google Scholar 

  23. Khoshouei, M., Radjainia, M., Baumeister, W. & Danev, R. Nat. Commun. 8, 16099 (2017).

    Article  CAS  Google Scholar 

  24. Barad, B.A. et al. Nat. Methods 12, 943–946 (2015).

    Article  CAS  Google Scholar 

  25. Herzik, M.A. Jr., Wu, M. & Lander, G.C. Setting up the Talos Arctica electron microscope and Gatan K2 direct detector for high-resolution cryogenic single-particle data acquisition. Protocol Exchange http://dx.doi.org/10.1038/protex.2017.108 (2017).

  26. Dubochet, J. et al. Q. Rev. Biophys. 21, 129–228 (1988).

    Article  CAS  Google Scholar 

  27. Grassucci, R.A., Taylor, D. & Frank, J. Nat. Protoc. 3, 330–339 (2008).

    Article  CAS  Google Scholar 

  28. Asadabad, M.A. & Eskandari, M.J. in Modern Electron Microscopy in Physical and Life Sciences. (eds. M. Janacek & R. Kral) (InTech, 2016).

  29. Voss, N.R., Yoshioka, C.K., Radermacher, M., Potter, C.S. & Carragher, B. J. Struct. Biol. 166, 205–213 (2009).

    Article  CAS  Google Scholar 

  30. Ogura, T., Iwasaki, K. & Sato, C. J. Struct. Biol. 143, 185–200 (2003).

    Article  CAS  Google Scholar 

  31. Roseman, A.M. J. Struct. Biol. 145, 91–99 (2004).

    Article  CAS  Google Scholar 

  32. Scheres, S.H. & Chen, S. Nat. Methods 9, 853–854 (2012).

    Article  CAS  Google Scholar 

  33. Chen, S. et al. Ultramicroscopy 135, 24–35 (2013).

    Article  CAS  Google Scholar 

  34. Punjani, A., Rubinstein, J.L., Fleet, D.J. & Brubaker, M.A. Nat. Methods 14, 290–296 (2017).

    Article  CAS  Google Scholar 

  35. Cardone, G., Heymann, J.B. & Steven, A.C. J. Struct. Biol. 184, 226–236 (2013).

    Article  Google Scholar 

  36. Herzik, M.A., Fraser, J.S. & Lander, G.C. A multi-model approach to assessing local and global cryo-EM map quality. Preprint at bioRxiv https://doi.org/10.1101/128561 (2017).

  37. Apweiler, R. et al. Nucleic Acids Res. 32, D115–D119 (2004).

    Article  CAS  Google Scholar 

  38. Wang, R.Y. et al. eLife 5, e17219 (2016).

    Article  Google Scholar 

  39. Chen, V.B. et al. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  40. Adams, P.D. et al. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J.-C. Ducom at The Scripps Research Institute High Performance Computing for computational support, B. Anderson at The Scripps Research Institute electron microscopy facility for microscope support, and M. Vos for advice and discussion regarding microscope alignments. We are grateful to Y. Cheng and Z. Yu (University of California, San Francisco) for kindly providing the 20S sample used in this study. M.A.H. is supported by a Helen Hay Whitney Foundation postdoctoral fellowship. G.C.L. is supported as a Searle Scholar and as a Pew Scholar, and by the US National Institutes of Health (NIH) grant DP2EB020402. Computational analyses of EM data were performed using shared instrumentation funded by NIH S10OD021634.

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Author notes

  1. Mark A Herzik and Mengyu Wu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA

    Mark A Herzik Jr, Mengyu Wu & Gabriel C Lander

Authors
  1. Mark A Herzik Jr
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  2. Mengyu Wu
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  3. Gabriel C Lander
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Contributions

M.A.H. and M.W. performed all cryo-EM experiments and analyses. M.A.H., M.W. and G.C.L. contributed to the experimental design and manuscript preparation.

Corresponding author

Correspondence to Gabriel C Lander.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Parallel illumination on a Talos Arctica TEM.

Image collected in diffraction mode at a distance of D 850 mm showing parallel illumination on a Talos Arctica TEM (2 condenser lens system). The objective aperture was brought into focus in the front focal plane of the diffraction lens using defocus – as evidenced by the crisp edges of the objective aperture (100 μm) – followed by spreading of the beam until the width of the gold powder diffraction rings were minimized. Gold powder diffraction rings are annotated. The inserted beam stop occludes the unscattered electron beam.

Supplementary Figure 2 Data collection, image processing and refinement of frozen-hydrated 20S proteasome collected using stage position.

(A) Typical low-mag image (LM-280x) of an individual Quantifoil UltrAUFoil® square with an acquisition target highlighted by a blue box. (B) Higher magnification (M-1250x) image of holes indicated by the blue box in panel (A). Exposure targets are indicated by green squares and the hole sacrificed for focusing is indicated by an orange square. (C) Representative high-magnification (SA-45000x), motion-corrected image of 20S proteasome in vitreous ice acquired using stage position for navigation. (D) 153,429 “particles” were isolated from aligned micrographs, Fourier binned 4 x 4, and subjected to reference-free 2D classification using RELION. Representative 2D class averages are displayed. The two right-most averages correspond to end-on views of the intact 20S (28-mer, left) and a 14-mer 20S subcomplex comprising seven and seven subunits (right). The “best” 106,581 particles were 3D auto-refined to yield a ~7.3 Å reconstruction. Refined particle coordinates were used for extraction of unbinned particles prior to 3D auto-refinement. Particles contributing to the final reconstruction at 3.32 Å resolution, were used for local estimation of defocus prior to re-extraction using a 512 box size. These particles yielded a 3.14 Å resolution reconstruction as estimated by gold-standard FSC. Attempts to further classify these particles or filter by particle alignment confidence did not improve the nominal FSC-reported resolution. Removal of particles collected with a defocus greater than 2 μm yielded the same resolution reconstruction as the full particle stack. For each reconstruction, the number of contributing particles and gold-standard FSC-reported resolution is listed below, respectively. (E) Euler distribution plot for the final reconstruction. (F) FSC curves calculated between the top-refined atomic model and the final particle stack (blue-line) or an unfiltered half map (green line). (G) FSC curves calculated between the top-refined atomic model and the final particle stack (blue-line) or a summed volume calculated from the top 10 refined models and the final map (orange line). (H) Worm representation with the ASU colored according to the per-residue C RMSD value (Å, the rest of the molecule is colored wheat). (I) Backbone atoms for the ASU from the top 10 refined atomic models shown in line representation. (J) Histogram of the per-residue C RMSD values calculated from the top 10 refined atomic models with the mean per-residue C RMSD value shown as a black vertical bar.

Supplementary Figure 3 Comparison of 20S proteasome EM density quality at ~3-Å resolution – 200 keV vs. 300 keV.

(Top row) EM density zoned 2 Å around the -sheet within the -subunit of the 20S proteasome from the Talos Arctica data set acquired using stage position (left panel, gray density, 106,581 particles), the EMDB entry 6287 (right panel, cyan density, 59,864 particles), as well as an overlay of each EM density (middle panel). For each panel, the best model refined against the Talos Arctica EM density is shown in wheat. (Bottom row) The same region of EM density from a reconstruction that used only particles from the stage position data set that had been imaged at ≤ 1.5 uM underfocus (3.19 Å resolution, light gray mesh, 52,501 particles).

Supplementary Figure 4 Ordered waters are conserved across 20S proteasome structures.

(A) EM density (gray mesh) from the 20S proteasome data set acquired using stage position zoned 20 Å around a set of three ordered water molecules (red spheres) placed using the program COOT and refined using Phenix. (B) PDB ID: 1YAR and the associated 2mFo-dFc electron density (gray mesh) zoomed to the same region as in panel A. (C) EM density (gray mesh) from the 20S proteasome EMDB entry 6287 with PDB ID:1YAR rigid-body fit and zoomed to the same region as in panel A. For each panel, putative hydrogen bonds are shown as dotted black lines.

Supplementary Figure 5 Data collection, image processing and refinement of frozen-hydrated 20S proteasome collected using image shift.

(A) Typical low-mag image (LM-280x) of an individual Quantifoil UltrAUFoil® square with an acquisition target highlighted by a blue box. (B) Higher magnification (M-2000x) image of holes indicated by the blue box in panel (A). Exposure targets are indicated by green squares and the hole sacrificed for focusing is indicated by an orange square. (C) Representative high-magnification (SA-45000x), motion-corrected image of 20S proteasome in vitreous ice acquired using image shift for navigation. (D) 111,184 “particles” were isolated from aligned micrographs, Fourier binned 4 x 4, and subjected to reference-free 2D classification using RELION. Representative 2D class averages are displayed. The “best” 96,254 particles were 3D auto-refined to yield a ~7.5 Å reconstruction. Refined particle coordinates were used for extraction of unbinned particles prior to 3D auto-refinement. Particles contributing to the final reconstruction, 3.32 Å resolution, were used for local estimation of defocus prior to re-extraction using a 512 box size. These particles yielded a 3.26 Å resolution reconstruction as estimated by gold-standard FSC. Attempts to further classify these particles did not improve the nominal FSC-reported resolution. For each reconstruction, the number of contributing particles and gold-standard FSC-reported resolution is listed below, respectively. (E) Euler distribution plot for the final reconstruction. (F) FSC curves calculated between the top-refined atomic model and the final particle stack (blue-line) or an unfiltered half map (green line). (G) FSC curves calculated between the top-refined atomic model and the final particle stack (blue-line) or a summed volume calculated from the top 10 refined models and the final map (orange line). (H) Worm representation with the ASU colored according to the per-residue C RMSD value (Å, the rest of the molecule is colored wheat). (I) Backbone atoms for the ASU from the top 10 refined atomic models shown in line representation. (J) Histogram of the per-residue C RMSD values calculated from the top 10 refined atomic models with the mean per-residue C RMSD value shown as a black vertical bar.

Supplementary Figure 6 Data collection, image processing and refinement of frozen-hydrated rabbit muscle aldolase collected using stage position.

(A) Representative high-magnification (SA-45000x), motion-corrected and dose-weighted image of aldolase in vitreous ice acquired using stage position for navigation. (B) CTF estimation fit (shown in blue) to experimental data and colored by CC (green, CC ≥90). Corresponding CTF estimates exhibit good fits to ~3 Å. (C) 1,009,341 particles were extracted from aligned micrographs, Fourier binned 4 x 4, and subjected to reference-free 2D classification using RELION. Representative 2D class averages are shown. Particles comprising the “best” class averages were 3D auto-refined to yield a ~7.5 Å reconstruction. Refined particle coordinates were used for local estimation of defocus prior to re-centering and extraction of particles, Fourier binned by 2 x 2, followed by 3D auto-refinement and no-alignment 3D classification. For each class, the number of contributing particles and percentage relative to total particles input to classification are listed below, respectively. Particles corresponding to the best-resolved classes were combined and 3D auto-refined to yield a ~3.6 Å reconstruction. Refined particle coordinates were re-centered and extract unbinned and refined once again to obtain a final reconstruction at ~2.6 Å resolution. (D) Euler distribution plot for the final reconstruction. (E) FSC curve calculated between the top-refined atomic model and the final particle stack (blue-line) or (F) a summed volume calculated from the top 10 refined models and the final map (orange line). (G) Worm representation with the ASU colored according to the per-residue C RMSD value (Å, the rest of the molecule is colored wheat). (H) Backbone atoms for the ASU from the top 10 refined atomic models shown in line representation. (I) Histogram of the per-residue C RMSD values calculated from the top 10 refined atomic models with the mean per-residue C RMSD value shown as a black vertical bar.

Supplementary Figure 7 Ordered waters are conserved across rabbit muscle aldolase structures.

(A) EM density (gray mesh) from the aldolase data set acquired using stage position zoned 15 Å around a set of four ordered water molecules (red spheres) placed using the program COOT and refined using Phenix. (B) PDB ID: 6ALD and the associated 2mFo-dFc electron density (gray mesh) zoomed to the same region as in panel A. For each panel, putative hydrogen bonds are shown as dotted black lines. Water molecules conserved between aldolase structures denoted with an asterisk (*).

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Notes 1–3

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Supplementary Protocol

Setting up the Talos Arctica electron microscope and Gatan K2 direct detector for high-resolution cryogenic single-particle data acquisition

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Herzik, M., Wu, M. & Lander, G. Achieving better-than-3-Å resolution by single-particle cryo-EM at 200 keV. Nat Methods 14, 1075–1078 (2017). https://doi.org/10.1038/nmeth.4461

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