bioRxiv2025
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