We have recently developed “monolayer purification” as a rapid and convenient technique to produce specimens of His-tagged proteins or macromolecular complexes for single particle electron microscopy (EM) without prior biochemical purification. Here, we introduce the “Affinity Grid”, a pre-fabricated EM grid featuring a dried lipid monolayer that contains Ni-NTA lipids (lipids functionalized with a Nickel-nitrilotriacetic acid group). The Affinity Grid, which can be stored for several months under ambient conditions, further simplifies and extends the use of monolayer purification. After characterizing the Affinity Grid, we used it to isolate, within minutes, ribosomal complexes from E. coli cell extracts containing His-tagged rpl3, the human homolog of the E. coli 50S subunit rplC. Depending on the way the sample was applied to the Affinity Grid, ribosomal complexes with or without associated mRNA could be prepared. Vitrified Affinity Grid specimens could be used to calculate three-dimensional reconstructions of the 50S ribosomal subunit as well as the 70S ribosome and 30S ribosomal subunit from images of the same sample. In addition, we established that Affinity Grids are stable for some time in the presence of glycerol and detergents. This feature allowed us to isolate His-tagged aquaporin-9 (AQP9) from detergent-solubilized membrane fractions of Sf9 insect cells. The Affinity Grid can thus be used to prepare single particle EM specimens of soluble complexes and membrane proteins.
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