Structural identification of endogenous Escherichia coli proteins from heterogeneous cryo-electron microscopy...

Single-particle cryo-electron microscopy (cryo-EM) can resolve multiple structurally distinct macromolecules (for example unintended host-derived proteins that remain during recombinant protein purification) within a heterogeneous specimen. Here, cryo-EM was initially performed to investigate an oligomeric form of recombinant human 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (OGFOD1) purified from Escherichia coli. However, image classification and 3D reconstruction revealed OGFOD1-incompatible particle populations. Combined cryo-EM and mass spectrometry identified these particles as three endogenous E. coli proteins: the bifunctional enzyme ArnA, guanosine triphosphate cyclohydrolase I (GTP cyclohydrolase I) and glycerol dehydrogenase (GlyDH). Their structures were reconstructed at overall resolutions of 3.24, 3.00 and 3.02 Å, respectively, revealing expected hexameric, decameric and octameric protein assemblies. Structural comparisons with previously reported crystallographic and cryo-EM structures showed that the overall oligomeric architectures and active-site configurations were highly conserved; however, local conformational differences were observed in specific regions. Despite substantial differences in total molecular mass, the three protein assemblies had comparable predicted hydrodynamic radii, which plausibly explained their partial coelution during size-exclusion chromatography. These results show that rigid, symmetric endogenous complexes can dominate heterogeneous cryo-EM datasets and highlight the value of systematic analysis of off-target particle populations.