Abstract
A density-modification procedure for improving maps from single-particle electron cryogenic microscopy (cryo-EM) is presented. The theoretical basis of the method is identical to that of maximum-likelihood density modification, previously used to improve maps from macromolecular X-ray crystallography. Key differences from applications in crystallography are that the errors in Fourier coefficients are largely in the phases in crystallography but in both phases and amplitudes in cryo-EM, and that half-maps with independent errors are available in cryo-EM. These differences lead to a distinct approach for combination of information from starting maps with information obtained in the density-modification process. The density-modification procedure was applied to a set of 104 datasets and improved map-model correlation and increased the visibility of details in many of the maps. The procedure requires two unmasked half-maps and a sequence file or other source of information on the volume of the macromolecule that has been imaged.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The source data for Figs. 1 and 2 are available as Excel worksheets. The spreadsheet and underlying data used to generate the figures in this work and examples of the density-modified maps presented in the figures are available at http://phenix-online.org/phenix_data/terwilliger/denmod_2020/.
References
Nogales, E. The development of cryo-EM into a mainstream structural biology technique. Nat. Methods 13, 24–27 (2016).
Marques, M. A., Purdy, M. D. & Yeager, M. CryoEM maps are full of potential. Curr. Opin. Struct. Biol. 58, 214–223 (2019).
Terwilliger, T. C., Adams, P. D., Afonine, P. V. & Sobolev, O. V. Cryo-EM map interpretation and protein model-building using iterative map segmentation. Protein Sci. 29, 87–99 (2019).
Wang, B. C. Resolution of phase ambiguity in macromolecular crystallography. Methods Enzymol. 115, 90–112 (1985).
Podjarny, A. D., Rees, B. & Urzhumtsev, A. G. in Crystallographic Methods and Protocols (eds Jones, C., Mulloy, B. & Sanderson, M. R.) 205–226 (Humana Press, 1996).
Cowtan, K. Recent developments in classical density modification. Acta Crystallogr. D. 66, 470–478 (2010).
Terwilliger, T. Maximum-likelihood density modification. Acta Crystallogr. D. 56, 965–972 (2000).
Scheres, S. H. A Bayesian view on cryo-EM structure determination. J. Mol. Biol. 415, 406–418 (2012).
Sindelar, C. V. & Grigorieff, N. Optimal noise reduction in 3D reconstructions of single particles using a volume-normalized filter. J. Struct. Biol. 180, 26–38 (2012).
Cheng, Y., Grigorieff, N., Penczek, P. A. & Walz, T. A primer to single-particle cryo-electron microscopy. Cell 161, 438–449 (2015).
Ramlaul, K., Palmer, C. M. & Aylett, C. H. S. A local agreement filtering algorithm for transmission EM reconstructions. J. Struct. Biol. 205, 30–40 (2019).
Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).
Cardone, G., Heymann, J. B. & Steven, A. C. One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J. Struct. Biol. 184, 226–236 (2013).
Spiegel, M., Duraisamy, A. K. & Schröder, G. F. Improving the visualization of cryo-EM density reconstructions. J. Struct. Biol. 191, 207–213 (2015).
Murshudov, G. N. in Methods in Enzymology Vol. 579 (ed. Crowther, R. A.) 277–305 (Academic Press, 2016).
Rosenthal, P. B. & Rubinstein, J. L. Validating maps from single particle electron cryomicroscopy. Curr. Opin. Struct. Biol. 34, 135–144 (2015).
Lawson, C. L. et al. EMDataBank.org: unified data resource for CryoEM. Nucleic Acids Res. 39, D456–D464 (2011).
Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).
Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).
Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).
Afanasyev, P. et al. Single-particle cryo-EM using alignment by classification (ABC): the structure of Lumbricus terrestris haemoglobin. IUCrJ 4, 678–694 (2017).
van Heel, M. & Schatz, M. Reassessing the revolution’s resolutions. Preprint at https://www.biorxiv.org/content/10.1101/224402v1 (2017).
van Heel, M. & Schatz, M. Fourier shell correlation threshold criteria. J. Struct. Biol. 151, 250–262 (2005).
Urzhumtsev, A., Afonine, P. V., Lunin, V. Y., Terwilliger, T. C. & Adams, P. D. Metrics for comparison of crystallographic maps. Acta Crystallogr. D. 70, 2593–2606 (2014).
Bartesaghi, A. et al. 2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor. Science 348, 1147 (2015).
Horst, B. G. et al. Allosteric activation of the nitric oxide receptor soluble guanylate cyclase mapped by cryo-electron microscopy. eLife 8, e50634 (2019).
Iudin, A., Korir, P. K., Salavert-Torres, J., Kleywegt, G. J. & Patwardhan, A. EMPIAR: a public archive for raw electron microscopy image data. Nat. Methods 13, 387–388 (2016).
Tang, G. et al. EMAN2: An extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).
Jakobi, A. J., Wilmanns, M. & Sachse, C. Model-based local density sharpening of cryo-EM maps. eLife 6, e27131 (2017).
Terwilliger, T. Improving macromolecular atomic models at moderate resolution by automated iterative model building, statistical density modification and refinement. Acta Crystallogr. D. 59, 1174–1182 (2003).
Skubak, P. et al. A new MR-SAD algorithm for the automatic building of protein models from low-resolution X-ray data and a poor starting model. IUCrJ 5, 166–171 (2018).
Bricogne, G. Geometric sources of redundancy in intensity data and their use for phase determination. Acta Crystallogr. A. 30, 395–405 (1974).
Abrahams, J. P. & Leslie, A. G. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D. 52, 30–42 (1996).
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D. 75, 861–877 (2019).
Masuda, T., Goto, F., Yoshihara, T. & Mikami, B. The universal mechanism for iron translocation to the ferroxidase site in ferritin, which is mediated by the well conserved transit site. Biochem. Biophys. Res. Commun. 400, 94–99 (2010).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D. 74, 531–544 (2018).
Terwilliger, T. C., Sobolev, O. V., Afonine, P. V. & Adams, P. D. Automated map sharpening by maximization of detail and connectivity. Acta Crystallogr. D. 74, 545–559 (2018).
Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D. 71, 136–153 (2015).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Terwilliger, T. Map-likelihood phasing. Acta Crystallogr. D. 57, 1763–1775 (2001).
Terwilliger, T. Reciprocal-space solvent flattening. Acta Crystallogr. D. 55, 1863–1871 (1999).
Scheres, S. H. Processing of structurally heterogeneous Cryo-EM Data in RELION. Methods Enzymol. 579, 125–157 (2016).
Shaikh, T. R., Hegerl, R. & Frank, J. An approach to examining model dependence in EM reconstructions using cross-validation. J. Struct. Biol. 142, 301–310 (2003).
Sousa, D. & Grigorieff, N. Ab initio resolution measurement for single particle structures. J. Struct. Biol. 157, 201–210 (2007).
Zhang, K. Y. J., Cowtan, K. & Main, P. Combining constraints for electron-density modification. Meth. Enzymol. 277, 53–64 (1997).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D. 66, 12–21 (2010).
Barad, B. A. et al. EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).
Park, E. & MacKinnon, R. Structure of the CLC-1 chloride channel from Homo sapiens. eLife 7, e36629 (2018).
Acknowledgements
This work was supported by the National Institutes of Health (grant no. GM063210 to P.D.A., R.J.R. and T.C.T. and grant no. R01-GM080139 to S.J.L.), the Wellcome Trust (grant no. 20947/Z/17/Z to R.J.R.) and the Phenix Industrial Consortium. This work was supported in part by the US Department of Energy under Contract No. DE-AC02-05CH11231 at the Lawrence Berkeley National Laboratory.
Author information
Authors and Affiliations
Contributions
S.J.L. carried out image processing of test datasets to evaluate varying reconstruction procedures. R.J.R. and T.C.T. contributed ideas on the form of errors in cryo-EM. P.V.A. developed tools for the testing infrastructure. T.C.T. developed the software for error analysis. P.D.A. and T.C.T. supervised the work.
Corresponding author
Ethics declarations
Competing Interests
The authors declare no competing interests.
Additional information
Peer review information Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Supplementary Information
Supplementary Figs. 1–9.
Source data
Source Data Fig. 1
Data used to generate Fig. 1AB
Source Data Fig. 2
Data used to generate Fig. 2AB
Rights and permissions
About this article
Cite this article
Terwilliger, T.C., Ludtke, S.J., Read, R.J. et al. Improvement of cryo-EM maps by density modification. Nat Methods 17, 923–927 (2020). https://doi.org/10.1038/s41592-020-0914-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41592-020-0914-9
This article is cited by
-
SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains
Nature (2024)
-
Structural basis of branching during RNA splicing
Nature Structural & Molecular Biology (2024)
-
Molecular details of ruthenium red pore block in TRPV channels
EMBO Reports (2024)
-
Multi-modal cryo-EM reveals trimers of protein A10 to form the palisade layer in poxvirus cores
Nature Structural & Molecular Biology (2024)
-
De novo design of pH-responsive self-assembling helical protein filaments
Nature Nanotechnology (2024)