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Cryo-EM structure of a methanogen nitrogenase–PII protein supercomplex

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Data availability

The single-particle cryo-EM maps and models have been deposited into the Protein Data Bank and Electron Microscopy Data Bank with the following accession codes: state 1 (D*KKD*) PDB 11UY and EMD-76071; state 2 (DKKD*) PDB 11SX and EMD-76025; state 3 (PII–DKKD*) PDB 11MY and EMD-75852; state 4 (supercomplex (C1 symmetry), (PII–DKKD–PII)3 PDB 9P1X and EMD-71144; state 4 (supercomplex (C3 symmetry), (PII–DKKD–PII)3 PDB 12AH and EMD-70378; state 5 (D#KKD*) PDB 11ZK and EMD-76217; and supercomplex (PII–DKKD–PII)3 from strictly anoxic grids PDB 37CY and EMD-78079. Source data are provided with this paper.

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Acknowledgements

We thank S. Brock, K. Basore and B. Readnour at the Washington University Center for Cellular Imaging and L. Wang, G. Hu and J. Kaminsky at the Laboratory for BioMolecular Structure (LBMS) at Brookhaven National Labs for cryo-EM data collection. We also thank R. Warmack (Yale University) for assistance with anaerobic grid preparation.

Funding

This work was supported by grants from the Department of Energy, Office of Basic Energy Sciences, DE-SC0020965 (E.A.) for structure determination; DE-SC0019226 (D.J.L.) for strain construction, protein purification and activity analyses; and DE-SC0018143 (B.B.) for mass spectrometry. The LBMS is supported by the Department of Energy, Office of Biological and Environmental Research (KP1607011). Funding for the Montana State Mass Spectrometry Facility (RRID: SCR_012482) used in this publication was made possible in part by the MJ Murdock Charitable Trust and the MSU office of the VPRED. We also acknowledge financial support from the Doisy Research Fund of the Edward A. Doisy Department of Biochemistry and Molecular Biology at Saint Louis University School of Medicine.

Author information

Author notes

  1. Ahmed Dhamad

    Present address: Department of Biological Sciences, Wasit University, Wasit, Iraq

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, USA

    Rajnandani Kashyap & Edwin Antony

  2. Department of Biological Sciences, University of Arkansas, Fayetteville, AR, USA

    Thomas M. Deere, Ahmed Dhamad, Melissa Chanderban & Daniel J. Lessner

  3. Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA

    Monika Tokmina-Lukaszewska & Brian Bothner

Authors

  1. Rajnandani Kashyap
  2. Thomas M. Deere
  3. Ahmed Dhamad
  4. Melissa Chanderban
  5. Monika Tokmina-Lukaszewska
  6. Brian Bothner
  7. Daniel J. Lessner
  8. Edwin Antony

Contributions

Conceptualization: E.A. and D.J.L. Methodology: R.K., T.M.D., A.D., M.C., M.T.-L. and B.B. Investigation: R.K., T.M.D., A.D., M.C. and M.T.-L. Visualization: R.K., T.M.D., E.A. and D.J.L. Funding acquisition: E.A., D.J.L. and B.B. Project administration: E.A. and D.J.L. Supervision: E.A., D.J.L and B.B. Writing—original draft: D.J.L., E.A. and R.K. Writing—review and editing: all authors.

Corresponding authors

Correspondence to Daniel J. Lessner or Edwin Antony.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Oliver Einsle, Mark Herzik Jr and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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 Coordination environment of metal clusters in NifDK from one half of unit 1 and identification of the homocitrate cofactor.

(A) Detailed view of the M-cluster coordination, emphasizing the key amino acid residues involved in ligation and stabilization of the cluster. (B) Detailed view of the P-cluster coordination, highlighting residues interacting with the iron-sulfur core. (C) Represents extracted ion chromatogram (EIC) for m/z = 205.0348 which corresponds to the calculated mass of homocitrate ion in negative mode. (D-E) Represents the fragmentation pattern of m/z = 205.0348 collected at two different collision energies: 10 eV and 15 eV, respectively. Annotated peaks represent identified fragments; underlined values represent diagnostic ions, specific only to the homocitrate molecule. m/z peaks denoted with gray diamonds in (D) represent signals from instrument background contaminants. (F) The ion structures and their m/z values are shown.

Extended Data Fig. 2 Structural features of the three NifDK heterotetramers in the supercomplex.

(A) Overlay of the three NifDK heterotetramers from the three units as shown in cylinder/stubs representation, demonstrating their overall structural similarity. (B-D) Electron density maps correspond to the M-cluster and P-cluster for unit 1, 2 and 3 respectively, with coordinating residues displayed for improved visualization.

Extended Data Fig. 3 Differential buried surface area between NifI2 within the NifI complex and NifDK across the supercomplex units.

(A) Unit 1, (B) Unit 2, and (C) Unit 3. Variations in the buried surface area at the interface are shown with the interface between the NifI complex and NifDK highlighted by dotted ovals in distinct colors for each unit. TSA denotes total surface area.

Extended Data Fig. 4 Comparison of ligand binding pockets and interactions between Glnk and the NifI complex from the supercomplex.

(A) Overlay of the Glnk structure (PDB: 2GNK) with the NifI complex, highlighting the three ligand binding pockets. (B) ADP-bound pocket 1 in the NifI complex (Unit 1) is overlaid with the ATP-bound pocket in the Glnk structure. Key residues that interact with their respective ligand are highlighted for both. For clarity, ADP and ATP are shown in separate panels within their respective binding pockets. (C) Ligand binding pocket 2 in the NifI complex bound to 2-oxoglutarate (2OG) is compared against the ATP-bound Glnk. Ligand coordinating residues are. (D) Ligand binding pocket 3 in the NifI complex supercomplex structure lacking a bound ligand is compared against the ATP-bound pocket in Glnk.

Extended Data Fig. 5 Overlay of the NifI complexes within the supercomplex.

(A) Overlay of all NifI complexes present in the supercomplex structure. (B) Overlay of the top and bottom NifI complexes from unit 1. (C) Overlay of the top and bottom NifI complexes from unit 2. (D) Overlay of the top and bottom NifI complexes from unit 3. The T-loops are marked.

Extended Data Fig. 6 Structural features of the D*KKD* complex (State-1).

(A) Overall structure of the D*KKD* complex. (B) Corresponding electron density map, with additional density from dynamic NifD chains (grey) not included in the modeled structure. (C) Carved electron density for the P-clusters observed on either side of the complex.

Extended Data Fig. 7 Structural features of the DKK complex (State-2).

(A) Overall structure of the DKK complex. (B) Corresponding electron density map, showing additional density from dynamic NifD chains (grey) not included in the modeled structure. Although some density for the P-cluster on the opposite side is visible, it was not modeled to ensure confidence in model building. (C) Carved electron density for the M- and the P-cluster observed in the complex.

Extended Data Fig. 8 Structural features of the PII-DKKD* complex (State-3).

(A) Overall structure of the PII-DKKD* complex. (B) Corresponding electron density map, showing additional density from dynamic NifD chains (grey) not included in the modeled structure. (C) Overlay of the one of the units from the supercomplex (green) with this structure (magenta), highlighting overall structural similarity and the missing NifD unit. (D) Carved electron density for the M- and P-clusters observed in the PII-DKKD* complex. (E) Close-up view of the PII complex extracted from this structure, with T-loops highlighted in blue. Individual NifI subunits are shown in distinct colors, consistent with the color scheme used throughout the manuscript. Bound ADP molecules associated with each subunit are depicted as sticks. (F) Carved density for ADP observed in the ligand-binding pockets for all three sites of the PII-DKKD* complex. (G) Overlay of one of the PII complex from the supercomplex (green) with this structure (magenta), showing overall similarity and differences in the loop region.

Extended Data Fig. 9 Mass photometry (MP) analysis of NifDK complexes.

MP of the native Strep-NifD pull down sample was measured at (A) 30 nM or (B) 10 nM. Higher concentrations were required to capture the supercomplex species (calculated mass 898 kDa). But, due to the high particle count for the major species (*), the mass interpretation is overestimated. The lower concentration measurement in panel B generates appropriate particle numbers for the major species and reflects the NifDK tetramer (calculated mass 225 kDa). However, the distribution is broad and likely reflects both PII-bound and PII-free NifDK tetramers. (C) The native Strep-NifD pull down sample was mixed with 2OG (0.1 mM) and ATP-Mg2+ (0.1 mM) and MP analysis shows presence of the NifDK tetramer along with transitions to a wide range of smaller sized complexes. (D) The PII-free NifDK sample was analyzed by MP. The NifDK tetramer is the major species along with both larger and smaller sized complexes.

Extended Data Fig. 10 Structural features of the PII-free D#KKD* complex (State-5).

(A) Overall structure of the PII-free D#KKD* complex. (B) Corresponding electron density map, showing additional density from dynamic NifD chains (eggplant) modelled in the structure. (C) Carved electron density for the M- and the P-cluster observed in the complex.

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Kashyap, R., Deere, T.M., Dhamad, A. et al. Cryo-EM structure of a methanogen nitrogenase–PII protein supercomplex. Nature (2026). https://doi.org/10.1038/s41586-026-11116-z

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