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TitleVisual exoproteomics of during anaerobic biomass-degradation identifies functional spirosomes.
Journal, issue, pagesbioRxiv, Year 2026
Publish dateMay 22, 2026
AuthorsMatthew P Agdanowski / Matthew J Kensil / Trevor H Moser / Ethan Humm / Yaneli I Guandique / Kayleigh Mason-Chalmers / Tracy Al-Set / Rachel R Ogorzalek Loo / James E Evans / Robert P Gunsalus / Joseph A Loo / Jose A Rodriguez
PubMed AbstractVisual proteomics enables the study of low-abundance proteins and identification of unknown complexes from heterogeneous samples by complementing high-resolution cryogenic electron microscopy (cryoEM) ...Visual proteomics enables the study of low-abundance proteins and identification of unknown complexes from heterogeneous samples by complementing high-resolution cryogenic electron microscopy (cryoEM) with external inputs on protein identity such as mass spectrometry. Using this approach, we interrogated the exoproteome of the anaerobic cellulose-degrading bacterium as it carried out biomass degradation. Mass spectrometry indicated a broad exoproteome composition, including cellulose degrading machinery CelA and CipA. A focus on large exoproteome assemblies revealed abundant protein filaments and pleomorphic vesicular structures. Analysis of the most abundant protein filaments yielded an ∼4 □ resolution native structure that, aided by mass spectrometry, modeling, and structural searching, was found to be the aldehyde-alcohol dehydrogenase (AdhE) spirosome. AdhE contained both NAD and Fe in their expected binding sites and biochemical and structural analyses of enriched spirosome preparations indicated they were functional. Altered NADH solution concentrations triggered conformational changes in the exoproteomic spirosomes, and the constituent AdhE remained capable of ethanol production. Although the basis for functional extracellular spirosome accumulation in live anaerobic cultures remains unclear, their abundance in crude exoproteomes suggests their presence could influence biomass fueled growth.
External linksbioRxiv / PubMed:42239438 / PubMed Central
MethodsEM (helical sym.)
Resolution4.07 Å
Structure data

EMDB-77065, PDB-13he:
CryoEM structure of AdhE spirosome from Clostridium thermocellum uncovered by visual proteomics.
Method: EM (helical sym.) / Resolution: 4.07 Å

Chemicals

ChemComp-FE:
Unknown entry

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM

Source
  • acetivibrio thermocellus dsm 1313 (bacteria)
KeywordsOXIDOREDUCTASE / Dehydrogenase / Spirosome / Filament

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