Recent progress in the development of affinity grids for cryo-EM typically employs genetic engineering of the protein sample such as histidine tagging, immobilized antibody capture, or non-selective immobilization <i>via</i> electrostatic interactions or Schiff base formation. We report a powerful and flexible method for affinity capture of target proteins for cryo-EM analysis that utilizes small molecule ligands as bait for concentrating human target proteins directly onto the grid surface for single particle reconstruction. This approach is demonstrated for human p97, captured using two different small molecule high affinity ligands of this AAA+ ATPase. Four electron density maps are revealed, each representing a p97 conformational state captured from solution, including a double-hexamer structure resolved to 3.6 Å.  These results demonstrate that non-covalent capture of protein targets on EM grids modified with high affinity ligands can enable structure elucidation of multiple configurational states of the target and potentially inform structure-based drug design campaigns.

Affinity capture of p97 with small molecule ligand bait reveals a 3.6Å double-hexamer structure by CryoEM

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