Warmack, R. A. | Search bioRxiv

bioRxiv2025

CryoEM-enabled visual proteomics reveals de novo structures of oligomeric protein complexes from Azotobacter vinelandii

Single particle cryoelectron microscopy (cryoEM) and cryoelectron tomography (cryoET) are powerful methods for unveiling unique and functionally relevant structural states. Aided by mass spectrometry and machine learning, they promise to facilitate the visual exploration of proteomes. Leveraging visual proteomics, we interrogate structures isolated from a complex cellular milieu by cryoEM to identify and classify molecular structures and complexes de novo. That approach determines the identity of six distinct oligomeric protein complexes from partially purified extracts of Azotobacter vinelandii using both anaerobic and aerobic cryoEM. Identification of the first unknown species, phosphoglucoisomerase (Pgi1), is achieved by comparing three automated model building programs: CryoID, DeepTracer, and ModelAngelo with or without a priori proteomics data. All three programs identify the Pgi1 protein, revealed to be in a new decameric state, as well as additional globular structures identified as glutamine synthetase (GlnA) and bacterioferritin (Bfr). Large filamentous assemblies are observed in tomograms reconstructed from cryoFIB milled lamellae of nitrogen-fixing A. vinelandii. Enrichment of these species from the cells by centrifugation allows for structure determination of three distinct filament types by helical reconstruction methods: the Type 6 Secretion System non-contractile sheath tube (TssC), a novel filamentous form of the soluble pyridine transhydrogenase (SthA), and the flagellar filament (FliC). The multimeric states of Pgi1 and SthA stand out in contrast to known crystallographic structures and offer a new structural framework from which to evaluate their activities. Overall, by allowing the study of near-native oligomeric protein states, cryoEM-enabled visual proteomics reveals novel structures that correspond to relevant species observed in situ. O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/636493v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@59bd1eorg.highwire.dtl.DTLVardef@18f0f36org.highwire.dtl.DTLVardef@12fe3a7org.highwire.dtl.DTLVardef@10aeda7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Anaerobic single particle cryoEM of nitrogenase

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 -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 -subunit reminiscent of that observed previously for the {Delta}nifB 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.

biochemistry↗