Format

Send to

Choose Destination
See comment in PubMed Commons below
Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4703-8. doi: 10.1073/pnas.0800867105. Epub 2008 Mar 17.

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

Author information

  • 1Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.

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.

PMID:
18347330
PMCID:
PMC2290746
DOI:
10.1073/pnas.0800867105
[PubMed - indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Support Center