National Institutes of Health/National Cancer Institute (NIH/NCI)
United States
Citation
Journal: ACS Nano / Year: 2021 Title: Affinity Capture of p97 with Small-Molecule Ligand Bait Reveals a 3.6 Å Double-Hexamer Cryoelectron Microscopy Structure. Authors: Md Rejaul Hoq / Frank S Vago / Kunpeng Li / Marina Kovaliov / Robert J Nicholas / Donna M Huryn / Peter Wipf / Wen Jiang / David H Thompson / 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 ...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.
History
Deposition
May 3, 2021
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Header (metadata) release
Jun 2, 2021
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Map release
Jun 2, 2021
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Update
Jun 9, 2021
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Current status
Jun 9, 2021
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Model: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 3.0 nm
Vitrification
Cryogen name: ETHANE / Chamber humidity: 80 % / Instrument: GATAN CRYOPLUNGE 3 / Details: Blot for 8 seconds before plunging..
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Electron microscopy
Microscope
FEI TITAN KRIOS
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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