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

ABSTRACT: 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 solutio

SUBMITTER: Hoq MR 

PROVIDER: S-EPMC11752781 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature