journal menu
![[Figure 3]](jb5071fig3mag.jpg)
|
|
Figure 3
Mechanisms underlying the persistence of ArnA through standard QC and its impact on cryo-EM analysis, with practical mitigation strategies. (a) His-tag purification allows endogenous ArnA to co-purify. Schematic illustration of partial overlap between ArnA and the intended target during size-exclusion chromatography (SEC). ArnA assembles into a compact hexamer (∼446 kDa), while the expected C. elegans KIF17–IFT70 complex (∼235 kDa, assuming a 2:1 stoichiometry) is predicted to adopt an extended, nonglobular conformation. Because SEC separation depends on hydrodynamic (Stokes) radius rather than MW alone, the extended KIF17–IFT70 complex can exhibit an increased apparent size and elute earlier than expected. When using a Superdex 200 Increase column, the resolving power of which decreases near its upper fractionation range (∼600 kDa), this effect can lead to partial elution overlap between the ArnA hexamer and the target complex, complicating separation by SEC alone. All panels are schematic illustrations and do not represent experimental data. (b) Cryo-EM readily identifies ArnA via its symmetric hexameric particles. Schematic comparison of particle alignability between rigid endogenous assemblies and flexible target complexes in single-particle cryo-EM. ArnA forms a rigid, symmetric hexamer that readily yields high-contrast projections and clean 2D class averages, facilitating rapid alignment and reconstruction. In contrast, the intended KIF17–IFT70 complex is low-yield, conformationally flexible and prone to partial disorder, resulting in heterogeneous particle views that are less competitive during alignment and classification. Even when present at lower abundance, rigid endogenous assemblies such as ArnA can therefore disproportionately dominate cryo-EM datasets. All panels are schematic illustrations and do not represent experimental data. (c) Suggested QC steps for low-yield recombinant proteins. Suggested QC checkpoints to minimize contaminant-driven cryo-EM reconstructions. Standard biochemical QC steps (SDS–PAGE, SEC and MS) may be insufficient to flag rigid endogenous contaminants present at low abundance. Early cryo-EM screening, particularly negative-stain EM and small-scale grid screening followed by 2D classification, can rapidly reveal homogeneous, symmetric particles indicative of contaminants such as ArnA. Incorporating low-background expression strains, orthogonal purification strategies and early image-based QC can substantially reduce the risk of allocating extensive cryo-EM resources to unintended targets. All panels are schematic illustrations and do not represent experimental data. |
![[Figure 3]](jb5071fig3.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
access
