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

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.

authors

Kelly DF,Dukovski D,Walz T

doi

10.1073/pnas.0800867105

subject

Has Abstract

pub_date

2008-03-25 00:00:00

pages

4703-8

issue

12

eissn

0027-8424

issn

1091-6490

pii

0800867105

journal_volume

105

pub_type

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