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Anaerobic single particle cryoEM of nitrogenase

Warmack, Rebeccah A. and Rees, Douglas C. (2022) Anaerobic single particle cryoEM of nitrogenase. . (Unpublished)

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The enzyme nitrogenase catalyzes the reduction of dinitrogen to ammonia during biological nitrogen fixation through a mechanism involving the ATP dependent interaction of two component proteins adopting multiple conformational states. To date, high resolution structural information has been provided by X-ray crystallography, which restricts the states that can be accessed to those that can be crystallized. Cryo-electron microscopy (cryoEM) presents a new opportunity for structural characterization of nitrogenase solution structures, and may yield new information on the mechanism of nitrogenase by revealing structures of transient or heterogeneous states. In this study, we present single particle cryoEM structures of the MoFe-nitrogenase endogenously isolated from Azotobacter vinelandii. To maintain the fully reduced cluster states of this oxygen sensitive protein, we prepared samples within an anaerobic chamber and employed specialized conditions to minimize partial disordering of the α-subunit at the air-water interface during freezing. Under these conditions, cryoEM structures of the as-isolated MoFe-protein and stabilized MoFe-protein-Fe-protein ADP-AlF4-complex were generally found to closely resemble their corresponding X-ray crystallographic structures. The cryoEM structures did reveal disordering in regions of the MoFe-protein α-subunit reminiscent of that observed previously for the ΔnifB MoFe-protein lacking the FeMo-cofactor, suggesting that this disorder may reflect functionally relevant dynamics, as well as the possibility of asymmetric binding of the Fe-protein to the MoFe-protein in solution. The methods presented here pave the way toward the capture and interrogation of turnover-relevant nitrogenase states by cryoEM.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription Paper
Warmack, Rebeccah A.0000-0002-9612-0511
Rees, Douglas C.0000-0003-4073-1185
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. The foundational contributions of Dr. Thomas Spatzal and Dr. Belinda Wenke to establishing the anaerobic Vitrobot system and conducting the original cryoEM structures of nitrogenase are gratefully acknowledged. We thank Dr. Jens Kaiser, Dr. Songye Chen, Ailiena Maggiolo, Przemyslaw Dutka, and Dr. James Howard for invaluable discussions. The generous support of the Beckman Institute for the Caltech CryoEM Resource Center was essential for the performance of this research. This work was funded by support from the Howard Hughes Medical Institute and NIH grant GM045162 (DCR) and NIH GM143836-01 (RAW). Data and materials availability. The single particle cryoEM maps and models have been deposited into the PDB and EMDB for release upon publication. The authors have declared no competing interest.
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Caltech Beckman InstituteUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Record Number:CaltechAUTHORS:20220607-424749000
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Official Citation:Anaerobic single particle cryoEM of nitrogenase Rebeccah A. Warmack, Douglas C. Rees bioRxiv 2022.06.04.494841; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:115048
Deposited By: George Porter
Deposited On:08 Jun 2022 15:10
Last Modified:08 Jun 2022 15:10

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