Affinity Capture of p97 with Small-Molecule Ligand Bait Reveals a 3.6 Å Double-Hexamer Cryoelectron Microscopy Structure

Md Rejaul Hoq, Frank S. Vago, Kunpeng Li, Marina Kovaliov, Robert J. Nicholas, Donna M. Huryn, Peter Wipf, Wen Jiang, David H. Thompson

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


Recent progress in the development of affinity grids for cryoelectron microscopy (cryo-EM) typically employs genetic engineering of the protein sample such as histidine or Spy tagging, immobilized antibody capture, or nonselective immobilization via electrostatic interactions or Schiff base formation. We report a powerful and flexible method for the 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 the noncovalent capture of protein targets on EM grids modified with high-Affinity ligands can enable the structure elucidation of multiple configurational states of the target and potentially inform structure-based drug design campaigns.

Original languageEnglish
Pages (from-to)8376-8385
Number of pages10
JournalACS Nano
Issue number5
StatePublished - 25 May 2021
Externally publishedYes

Bibliographical note

Funding Information:
Support from the Purdue Center for Cancer Research Special Project funds (CCSG CA23168) and the Indiana CTSI Grant (UL1TR001108) are gratefully acknowledged. Support of the small-molecule ligand synthesis efforts in Prof. Huryn’s lab by the National Cancer Institute, National Institutes of Health, under Chemical Biology Consortium contract no. HHSN261200800001E, agreement no. 29XS127TO15 is also noted. We also thank Prof. Tsui-Fen Chou (UCLA) for the gift of the plasmid containing the p97 gene and Stacie Bulfer (UCSF) for performing the IC assays. 50

Publisher Copyright:
© 2021 American Chemical Society.


  • AAA+ ATPase
  • affinity capture
  • affinity lipid monolayer
  • multiple hexameric protein states
  • p97
  • protein-inhibitor complex
  • single-particle reconstruction


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