Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins

Nature Protocols (2024)Cite this article

Subjects

Abstract

Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments.

Key points

  • Structural analysis of air-sensitive enzymes requires that strictly anaerobic conditions be maintained during sample preparation and analysis. In this protocol, a customized vacuum manifold is used in the purification of nitrogenases from Azotobacter vinelandii and a custom anaerobic chamber is used for protein preparation for cryo-electron microscopy.

  • An internal oxygen probe made by genetically tagging the nitrogenase Fe protein with the far-red fluorescent protein mPlum can be expressed to report on oxygen exposure.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overview of the procedure.
Fig. 2: Custom vacuum-manifold setup.
Fig. 3: Infrastructure for anaerobic single-particle cryoEM grid preparation.
Fig. 4: Overview of anaerobic cell lysis.
Fig. 5: Purifications of nitrogenase proteins from the wild-type and mPlum-tagged strains.
Fig. 6: Potential pitfalls or causes for troubleshooting.
Fig. 7: CryoEM structures of the nitrogenase Fe protein, MoFe protein and ADP–AlF4− stabilized complex states.

Similar content being viewed by others

Data availability

The single-particle cryoEM maps and models have been deposited into the PDB and Electron Microscopy Data Bank (EMDB) for release upon publication. Datasets been deposited with the following PDB and EMDB codes: 8TC3, EMD-41151 (Fe protein, mPlum tagged); 8DBY, EMD-27317 (MoFe protein on ultrathin carbon); 8DFC, EMD-27404 (ADP–AlF4− stabilized MoFe protein:Fe protein 1:1 complex); 8DFD, EMD-27405 (ADP–AlF4− stabilized MoFe protein:Fe protein 2:1 complex). All other data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

References

  1. Thamdrup, B. New pathways and processes in the global nitrogen cycle. Ann. Rev. Ecol. Evol. Syst. 43, 407–428 (2012).

    Article  Google Scholar 

  2. Thorneley, R. & Lowe, D. Molybdenum enzymes. Met. Ions Biol. 7, 221–284 (1985).

    CAS  Google Scholar 

  3. Holland, P. L. Introduction: reactivity of nitrogen from the ground to the atmosphere. Chem. Rev. 120, 4919–4920 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gallon, J. R. The oxygen sensitivity of nitrogenase: a problem for biochemists and micro-organisms. Trends Biochem. Sci. 6, 19–23 (1981).

    Article  CAS  Google Scholar 

  5. Lowe, D. & Thorneley, R. N. The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution. Biochem. J. 224, 895–901 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Buscagan, T. M., Kaiser, J. T. & Rees, D. C. Selenocyanate derived Se-incorporation into the nitrogenase Fe protein cluster. eLife 11, e79311 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Henthorn, J. T. et al. Localized electronic structure of nitrogenase FeMoco revealed by Selenium K-edge high resolution X-ray absorption spectroscopy. J. Am. Chem. Soc. 141, 13676–13688 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Spatzal, T., Perez, K. A., Howard, J. B. & Rees, D. C. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor. eLife 4, e11620 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Sippel, D. et al. A bound reaction intermediate sheds light on the mechanism of nitrogenase. Science 359, 1484–1489 (2018).

    Article  CAS  PubMed  Google Scholar 

  10. Buscagan, T. M., Perez, K. A., Maggiolo, A. O., Rees, D. C. & Spatzal, T. Structural characterization of two CO molecules bound to the nitrogenase active site. Angew. Chem. Int. Ed. 60, 5704–5707 (2021).

    Article  CAS  Google Scholar 

  11. Spatzal, T., Perez, K. A., Einsle, O., Howard, J. B. & Rees, D. C. Ligand binding to the FeMo-cofactor: structures of CO-bound and reactivated nitrogenase. Science 345, 1620–1623 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Einsle, O. & Rees, D. C. Structural enzymology of nitrogenase enzymes. Chem. Rev. 120, 4969–5004 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Nakane, T. et al. Single-particle cryo-EM at atomic resolution. Nature 587, 152–156 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Yip, K. M., Fischer, N., Paknia, E., Chari, A. & Stark, H. Atomic-resolution protein structure determination by cryo-EM. Nature 587, 157–161 (2020).

    Article  CAS  PubMed  Google Scholar 

  15. Warmack, R. A. et al. Structural consequences of turnover-induced homocitrate loss in nitrogenase. Nat. Commun. 14, 1091 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Rutledge, H. L., Cook, B. D., Nguyen, H. P. M., Herzik, M. A. & Tezcan, F. A. Structures of the nitrogenase complex prepared under catalytic turnover conditions. Science 377, 865–869 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Warmack, R. A. & Rees, D. C. Nitrogenase beyond the resting state: a structural perspective. Molecules 28, 7952 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shriver, D. F. & Drezdzon, M. A. The Manipulation of Air-Sensitive Compounds 2nd edn (Wiley, 1986).

  19. Lee, C. C., Ribbe, M. W. & Hu, Y. Purification of nitrogenase proteins. Methods Mol. Biol. 1876, 111–124 (2019).

    Article  CAS  PubMed  Google Scholar 

  20. Wiig, J. A., Lee, C. C., Fay, A. W., Hu, Y. & Ribbe, M. W. Purification of nitrogenase proteins. Methods Mol. Biol. 766, 93–103 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Jiménez-Vicente, E. et al. Application of affinity purification methods for analysis of the nitrogenase system from Azotobacter vinelandii. Methods Enzymol. 613, 231–255 (2018).

    Article  PubMed  Google Scholar 

  22. Spatzal, T. et al. Evidence for interstitial carbon in nitrogenase FeMo cofactor. Science 334, 940 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kim, J., Woo, D. & Rees, D. C. X-ray crystal structure of the nitrogenase molybdenum–iron protein from Clostridium pasteurianum at 3.0-A resolution. Biochemistry 32, 7104–7115 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. Mayer, S. M., Lawson, D. M., Gormal, C. A., Roe, S. M. & Smith, B. E. New insights into structure–function relationships in nitrogenase: a 1.6 A resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein. J. Mol. Biol. 292, 871–891 (1999).

    Article  CAS  PubMed  Google Scholar 

  25. Dos Santos, P. C. Molecular biology and genetic engineering in nitrogen fixation. Methods Mol. Biol. 766, 81–92 (2011).

    Article  PubMed  Google Scholar 

  26. Dos Santos, P. C. Genomic manipulations of the diazotroph Azotobacter vinelandii. Methods Mol. Biol. 1876, 91–109 (2019).

    Article  PubMed  Google Scholar 

  27. Echavarri-Erasun, C., Arragain, S. & Rubio, L. M. Purification of O2-sensitive metalloproteins. Methods Mol. Biol. 1122, 5–18 (2014).

    Article  CAS  PubMed  Google Scholar 

  28. Uchendu, S. N., Rafalowski, A., Cohn, E. F., Davoren, L. W. & Taylor, E. A. Anaerobic protein purification and kinetic analysis via oxygen electrode for studying DesB dioxygenase activity and inhibition. J. Vis. Exp. https://doi.org/10.3791/58307 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Uzarski, J. S., DiVito, M. D., Wertheim, J. A. & Miller, W. M. Essential design considerations for the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds. Biomaterials 129, 163–175 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Moore, M. M., Oteng-Pabi, S. K., Pandelieva, A. T., Mayo, S. L. & Chica, R. A. Recovery of red fluorescent protein chromophore maturation deficiency through rational design. PLoS ONE 7, e52463 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Linkerhägner, K. & Oelze, J. Cellular ATP levels and nitrogenase switchoff upon oxygen stress in chemostat cultures of Azotobacter vinelandii. J. Bacteriol. 177, 5289–5293 (1995).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Wenke, B. B., Arias, R. J. & Spatzal, T. Crystallization of nitrogenase proteins. Methods Mol. Biol. 1876, 155–165 (2019).

    Article  CAS  PubMed  Google Scholar 

  34. Wenke, B. B. The Many Roles of the Nitrogenase Iron Protein PhD thesis, California Institute of Technology (2019).

  35. Chen, J., Noble, A. J., Kang, J. Y. & Darst, S. A. Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: bacterial RNA polymerase and CHAPSO. J. Struct. Biol. X. https://doi.org/10.1016/j.yjsbx.2019.100005 (2019).

  36. Noble, A. J. et al. Routine single particle CryoEM sample and grid characterization by tomography. eLife https://doi.org/10.7554/eLife.34257 (2018).

  37. Noble, A. J. et al. Reducing effects of particle adsorption to the air-water interface in cryo-EM. Nat. Methods 15, 793–795 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Passmore, L. A. & Russo, C. J. Specimen preparation for high-resolution cryo-EM. Methods Enzymol. 579, 51–86 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wagner, A. O. et al. Medium preparation for the cultivation of microorganisms under strictly anaerobic/anoxic conditions. J. Vis. Exp. https://doi.org/10.3791/60155 (2019).

  40. Lambertz, C. et al. O2 reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase. J. Biol. Chem. 286, 40614–40623 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gillman, C., Nicolas, W. J., Martynowycz, M. W. & Gonen, T. Design and implementation of suspended drop crystallization. Preprint at bioRxiv https://doi.org/10.1101/2023.03.28.534639 (2023).

  42. Cherrier, M. V. et al. Oxygen-sensitive metalloprotein structure determination by cryo-electron microscopy. Biomolecules https://doi.org/10.3390/biom12030441 (2022).

  43. Tivol, W. F., Briegel, A. & Jensen, G. J. An improved cryogen for plunge freezing. Microsc. Microanal. 14, 375–379 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Schmidt, F. V. et al. Structural insights into the iron nitrogenase complex. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-023-01124-2 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Fajardo, A. S. et al. Structural insights into the mechanism of the radical SAM carbide synthase NifB, a key nitrogenase cofactor maturating enzyme. J. Am. Chem. Soc. 142, 11006–11012 (2020).

    Article  CAS  PubMed  Google Scholar 

  46. Kang, W. et al. X-Ray crystallographic analysis of NifB with a full complement of clusters: structural insights into the radical SAM-dependent carbide insertion during nitrogenase cofactor assembly. Angew. Chem. Int. Ed. 60, 2364–2370 (2021).

    Article  CAS  Google Scholar 

  47. Ferreira, C. M. H., Pinto, I. S. S., Soares, E. V. & Soares, H. M. V. M. (Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions—a review. RSC Adv. 5, 30989–31003 (2015).

    Article  CAS  Google Scholar 

  48. Scheller, K. H. et al. Metal ion/buffer interactions. Eur. J. Biochem. 107, 455–466 (1980).

    Article  CAS  PubMed  Google Scholar 

  49. Zawisza, I., Rózga, M., Poznański, J. & Bal, W. Cu(II) complex formation by ACES buffer. J. Inorg. Biochem. 129, 58–61 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Wolle, D., Kim, C., Dean, D. & Howard, J. B. Ionic interactions in the nitrogenase complex. Properties of Fe-protein-containing substitutions for Arg-100. J. Biol. Chem. 267, 3667–3673 (1992).

    Article  CAS  PubMed  Google Scholar 

  51. Robinson, A. C., Burgess, B. K. & Dean, D. R. Activity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster. J. Bacteriol. 166, 180–186 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Schlenk, W. & Thal, A. Über metallketyle, eine grosse klasse von verbindungen mit dreiwertigem kohlenstoff II. Ber. Dtsch. Chem. Ges. 46, 2840–2854 (1913).

    Article  Google Scholar 

  53. Dilworth, M. J. Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum. Biochim. Biophys. Acta 127, 285–294 (1966).

    Article  CAS  PubMed  Google Scholar 

  54. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  55. Thompson, R. F., Iadanza, M. G., Hesketh, E. L., Rawson, S. & Ranson, N. A. Collection, pre-processing and on-the-fly analysis of data for high-resolution, single-particle cryo-electron microscopy. Nat. Protoc. 14, 100–118 (2019).

    Article  CAS  PubMed  Google Scholar 

  56. Keable, S. M. et al. Structural characterization of the P(1+) intermediate state of the P-cluster of nitrogenase. J. Biol. Chem. 293, 9629–9635 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Danyal, K. et al. Negative cooperativity in the nitrogenase Fe protein electron delivery cycle. Proc. Natl Acad. Sci. USA 113, E5783–E5791 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Vénien-Bryan, C. & Fernandes, C. A. H. Overview of membrane protein sample preparation for single-particle cryo-electron microscopy analysis. Int. J. Mol. Sci. https://doi.org/10.3390/ijms241914785 (2023).

Download references

Acknowledgements

This work was funded by support from the Howard Hughes Medical Institute (D.C.R.), National Institutes of Health grants GM045162 (D.C.R.) and GM143836-01 (R.A.W.). The foundational contributions of J. Howard to establishing the anaerobic methodology within the laboratory are gratefully acknowledged. We thank J. Kaiser, S. Chen, A. Maggiolo and N. Siladke for their invaluable discussions. Azotobacter vinelandii DJ54 strain was a kind gift from D. Dean. The generous support of the Beckman Institute for the Caltech CryoEM Resource Center was essential for the performance of this research.

Author information

Authors and Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA

    Rebeccah A. Warmack, Belinda B. Wenke, Thomas Spatzal & Douglas C. Rees

  2. Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA

    Rebeccah A. Warmack & Douglas C. Rees

Authors
  1. Rebeccah A. Warmack
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Belinda B. Wenke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas Spatzal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Douglas C. Rees
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.S. and B.B.W. constructed and installed equipment. T.S., B.B.W. and R.A.W. established protocols. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Rebeccah A. Warmack or Douglas C. Rees.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Related links

Key reference using this protocol

Warmack, R. A. et al. Nat. Commun. 14, 1091 (2023): https://doi.org/10.1038/s41467-023-36636-4

Key data used in this protocol

Warmack, R. A. et al. Nat. Commun. 14, 1091 (2023): https://doi.org/10.1038/s41467-023-36636-4

Supplementary information

Source data

Source Data Fig. 6

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Warmack, R.A., Wenke, B.B., Spatzal, T. et al. Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00973-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41596-024-00973-5

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing