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spinqualitylabelsall models Cryo-EM reconstruction of ArnA obtained from the dataset. Ab initio reconstruction produced a 3-fold-symmetric map corresponding to the ArnA hexamer at 3.23Å (PDB-ID 21ak). The density is consistent with previously reported ArnA architectures, confirming that the dominant particles in the dataset correspond to ArnA contaminant, rather than the intended target complex. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Cryo-EM reveals ArnA contamination during purification of a ciliary protein complex Xuguang Jiang, Masahide Kikkawa Molecular Tour Protein purification does not always yield what it seems. While determining the cryo-EM structure of a soluble KIF17-IFT70 ciliary protein complex, we found that a minor bacterial contaminant dominated the structural dataset. SDS-PAGE, size-exclusion chromatography and mass spectrometry supported the presence of the intended proteins and gave little warning of a major contaminant. Cryo-EM instead revealed ArnA. Explore the structure: Render separately 21ak, (this study) and 9v5h (Caliseki et al.) or compare both structures superimposed. It is also interesting to . Fig. 1. Biochemical QC was misleading: ArnA (~74 kDa) overlaps with KIF17 (~79 kDa) and IFT70 (~76 kDa) on SDS-PAGE. ArnA peptides were recognized only retrospectively; contrast adjustment also reveals a faint ~74 kDa band (right panel) consistent with ArnA. Why ArnA wins in cryo-EM. ArnA forms a stable, highly symmetric hexamer that produces well-defined particle views and aligns efficiently during single-particle analysis. The intended KIF17-IFT70 complex was low-yield, flexible and prone to aggregation. Thus, even a small amount of ArnA could contribute a disproportionate number of high-quality particles and dominate the final reconstruction. The map and atomic model closely match previously determined ArnA structure. Cryo-EM reveals what biochemical QC missed. Micrographs contained aggregates together with dispersed, homogeneous particles, and 2D classification produced unusually clean threefold-symmetric views. Ab initio reconstruction identified the particles as the ArnA hexamer (Fig. 2); D3 refinement yielded a 3.23 Å reconstruction. No convincing KIF17-IFT70 classes were found. The close agreement with the previously reported ArnA structure made the contaminant unambiguous. Fig. 2. Comparison of cryo-EM maps. The map from this study closely matches ArnA reported by Caliseki et al., showing that the dominant reconstruction is ArnA rather than the intended KIF17-IFT70 ciliary protein complex. Practical lesson. Biochemical abundance and structural visibility are not equivalent. Rigid endogenous assemblies such as ArnA hexamers may remain inconspicuous during purification yet become immediately prominent by electron microscopy. Early negative-stain or cryo-EM screening, careful inspection of 2D classes, and awareness of recurrent host-derived contaminants can prevent substantial microscope time being spent on unintended targets. Take-home message for the I3DC. ArnA (PDB 21AK; EMD-67448) is both an atomic structure and a methodological warning: in cryo-EM, structural “fitness” for particle alignment can matter more than biochemical abundance. The symmetry and rigidity that make ArnA an excellent cryo-EM specimen are also what make it a troublesome contaminant. (PDB 21AK; EMD-67448) References