The Affinity Grid: a pre-fabricated EM grid for monolayer purification.
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 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 Escherichia coli cell extracts containing His-tagged rpl3, the human homolog of the E. coli 50 S subunit rplC. Ribosomal complexes with or without associated mRNA could be prepared depending on the way the sample was applied to the Affinity Grid . Vitrified Affinity Grid specimens could be used to calculate three-dimensional reconstructions of the 50 S ribosomal subunit as well as the 70 S ribosome and 30 S ribosomal subunit from images of the same sample. We established that Affinity Grids are stable for some time in the presence of glycerol and detergents, which 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.
Kelly
Deborah F
DF
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Abeyrathne
Priyanka D
PD
Dukovski
Danijela
D
Walz
Thomas
T
D020346:Aquaporins / Q000302:isolation & purification; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D046911:Macromolecular Substances / Q000302:isolation & purification*; D008854:Microscopy, Electron / Q000295:instrumentation* / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D021122:Protein Subunits / Q000302:isolation & purification; D011990:Receptors, Transferrin / Q000302:isolation & purification / Q000648:ultrastructure; D000090822:Ribosomal Protein L3; D012270:Ribosomes / Q000737:chemistry / Q000648:ultrastructure; D014168:Transferrin / Q000302:isolation & purification / Q000648:ultrastructure
D020346:Aquaporins; D008055:Lipids; D046911:Macromolecular Substances; D021122:Protein Subunits; C000716547:RPL3 protein, human; D011990:Receptors, Transferrin; D000090822:Ribosomal Protein L3; D014168:Transferrin