Monolayer purification: a rapid method for isolating protein complexes for single-particle electron microscopy.
Visualizing macromolecular complexes by single-particle electron microscopy (EM) entails stringent biochemical purification, specimen preparation, low-dose imaging, and 3D image reconstruction. Here, we introduce the "monolayer purification" method, which employs nickel-nitrilotriacetic acid (Ni-NTA) functionalized lipids for simultaneously purifying His-tagged complexes directly from cell lysates while producing specimens suitable for single-particle EM. The method was established by using monolayers containing Ni-NTA lipid to specifically adsorb His-tagged transferrin-transferrin receptor (Tf-TfR) complexes from insect and mammalian cell extracts. The specificity and sensitivity of the method could be improved by adding imidazole to the extracts. The monolayer-purified Tf-TfR samples could be vitrified and used to calculate a 3D reconstruction of the complex. Monolayer purification was then used to rapidly isolate ribosomal complexes from bacteria by overexpressing a single His-tagged ribosomal subunit. The resulting monolayer samples allowed calculation of a cryo-EM 3D reconstruction of the Escherichia coli 50S ribosomal subunit.
Kelly
Deborah F
DF
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Dukovski
Danijela
D
Walz
Thomas
T
D000327:Adsorption; D000818:Animals; D002457:Cell Extracts; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D020285:Cryoelectron Microscopy / Q000379:methods*; D006639:Histidine / Q000378:metabolism; D050356:Lipid Metabolism; D046912:Multiprotein Complexes / Q000302:isolation & purification* / Q000648:ultrastructure*; D009532:Nickel; D009571:Nitrilotriacetic Acid / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism / Q000648:ultrastructure; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000648:ultrastructure; D014168:Transferrin / Q000378:metabolism / Q000648:ultrastructure
D002457:Cell Extracts; C471213:His-His-His-His-His-His; D046912:Multiprotein Complexes; D009842:Oligopeptides; D011990:Receptors, Transferrin; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; D014168:Transferrin; D006639:Histidine; D009532:Nickel; D009571:Nitrilotriacetic Acid