Monolayer purification: A rapid method for isolating protein complexes for single-particle electron microscopy

Deborah F. Kelly, Danijela Dukovski, Thomas Walz

Research output: Contribution to journalArticlepeer-review

50 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4703-4708
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number12
DOIs
StatePublished - Mar 25 2008

All Science Journal Classification (ASJC) codes

  • General

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