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 journalArticle

49 Citations (Scopus)

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

Fingerprint

Ribosome Subunits
Electron Microscopy
Transferrin Receptors
Transferrin
Macromolecular Substances
Cryoelectron Microscopy
Lipids
Proteins
Computer-Assisted Image Processing
Cell Extracts
Insects
Escherichia coli
Bacteria
Sensitivity and Specificity
nickel nitrilotriacetic acid
imidazole

All Science Journal Classification (ASJC) codes

  • General

Cite this

@article{7e300b67089a4268ae279eb377b153f5,
title = "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.",
author = "Kelly, {Deborah F.} and Danijela Dukovski and Thomas Walz",
year = "2008",
month = "3",
day = "25",
doi = "10.1073/pnas.0800867105",
language = "English (US)",
volume = "105",
pages = "4703--4708",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "12",

}

Monolayer purification : A rapid method for isolating protein complexes for single-particle electron microscopy. / Kelly, Deborah F.; Dukovski, Danijela; Walz, Thomas.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 12, 25.03.2008, p. 4703-4708.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Monolayer purification

T2 - A rapid method for isolating protein complexes for single-particle electron microscopy

AU - Kelly, Deborah F.

AU - Dukovski, Danijela

AU - Walz, Thomas

PY - 2008/3/25

Y1 - 2008/3/25

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=42449146663&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=42449146663&partnerID=8YFLogxK

U2 - 10.1073/pnas.0800867105

DO - 10.1073/pnas.0800867105

M3 - Article

C2 - 18347330

AN - SCOPUS:42449146663

VL - 105

SP - 4703

EP - 4708

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 12

ER -