Strategy for the use of affinity grids to prepare non-his-tagged macromolecular complexes for single-particle electron microscopy

Deborah F. Kelly, Danijela Dukovski, Thomas Walz

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Affinity Grids are electron microscopy (EM) grids with a pre-deposited lipid monolayer containing functionalized nickel-nitrilotriacetic acid lipids. Affinity Grids can be used to prepare His-tagged proteins for single-particle EM from impure solutions or even directly from cell extracts. Here, we introduce the concept of His-tagged adaptor molecules, which eliminate the need for the target protein or complex to be His-tagged. The use of His-tagged protein A as adaptor molecule allows Affinity Grids to be used for the preparation of virtually any protein or complex provided that a specific antibody is available or can be raised against the target protein. The principle is that the Affinity Grid is coated with a specific antibody that is recruited to the grid by His-tagged protein A. The antibody-decorated Affinity Grid can then be used to isolate the target protein directly from a cell extract. We first established this approach by preparing negatively stained specimens of both native ribosomal complexes and ribosomal complexes carrying different purification tags directly from HEK-293T cell extract. We then used the His-tagged protein A/antibody strategy to isolate RNA polymerase II, still bound to native DNA, from HEK-293T cell extract, allowing us to calculate a 25-Å-resolution density map by single-particle cryo-EM.

Original languageEnglish (US)
Pages (from-to)675-681
Number of pages7
JournalJournal of Molecular Biology
Volume400
Issue number4
DOIs
StatePublished - Jul 1 2010

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Macromolecular Substances
Electron Microscopy
Cell Extracts
Staphylococcal Protein A
HEK293 Cells
Proteins
Antibodies
Cryoelectron Microscopy
Lipids
Antibody Affinity
RNA Polymerase II
DNA

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Cite this

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Strategy for the use of affinity grids to prepare non-his-tagged macromolecular complexes for single-particle electron microscopy. / Kelly, Deborah F.; Dukovski, Danijela; Walz, Thomas.

In: Journal of Molecular Biology, Vol. 400, No. 4, 01.07.2010, p. 675-681.

Research output: Contribution to journalArticle

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