Strategy for the use of affinity grids to prepare non-His-tagged macromolecular complexes for single-particle electron microscopy.
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-A-resolution density map by single-particle cryo-EM.
Kelly
Deborah F
DF
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Dukovski
Danijela
D
Walz
Thomas
T
D000906:Antibodies / Q000378:metabolism; D002457:Cell Extracts; D002460:Cell Line; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods*; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism / Q000648:ultrastructure; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure; D013048:Specimen Handling / Q000379:methods*
D000906:Antibodies; D002457:Cell Extracts; D046911:Macromolecular Substances; D012319:RNA Polymerase II