The Affinity Grid: A Pre-fabricated EM Grid for Monolayer Purification

doi:10.1016/j.jmb.2008.07.023

Journal of Molecular Biology

Volume 382, Issue 2, 3 October 2008, Pages 423-433

https://doi.org/10.1016/j.jmb.2008.07.023 Get rights and content

Abstract

We have recently developed monolayer purification as a rapid and convenient technique to produce specimens of His-tagged proteins or macromolecular complexes for single-particle electron microscopy (EM) without biochemical purification. Here, we introduce the Affinity Grid, a pre-fabricated EM grid featuring a dried lipid monolayer that contains Ni-NTA lipids (lipids functionalized with a nickel-nitrilotriacetic acid group). The Affinity Grid, which can be stored for several months under ambient conditions, further simplifies and extends the use of monolayer purification. After characterizing the Affinity Grid, we used it to isolate, within minutes, ribosomal complexes from Escherichia coli cell extracts containing His-tagged rpl3, the human homolog of the E. coli 50 S subunit rplC. Ribosomal complexes with or without associated mRNA could be prepared depending on the way the sample was applied to the Affinity Grid . Vitrified Affinity Grid specimens could be used to calculate three-dimensional reconstructions of the 50 S ribosomal subunit as well as the 70 S ribosome and 30 S ribosomal subunit from images of the same sample. We established that Affinity Grids are stable for some time in the presence of glycerol and detergents, which allowed us to isolate His-tagged aquaporin-9 (AQP9) from detergent-solubilized membrane fractions of Sf9 insect cells. The Affinity Grid can thus be used to prepare single-particle EM specimens of soluble complexes and membrane proteins.

Introduction

The biochemical purification of proteins and macromolecular complexes has become the rate-limiting step in structure determination by single-particle cryo-electron microscopy (EM). A protocol for the purification of a recombinant protein for structural studies typically consists of an affinity chromatography step followed by size-exclusion chromatography. Such a two-step purification scheme requires an appreciable amount of protein, which is not always achievable, and takes several hours, during which time biological complexes can disintegrate. We have recently introduced monolayer purification, a technique that combines protein purification with specimen preparation, as a rapid and simple way to prepare specimens suitable for single-particle EM, requiring only low expression levels or low concentrations of His-tagged protein.¹ In the monolayer purification technique, the extract of cells expressing a His-tagged protein, which can be a subunit of a larger assembly, is overlaid with a lipid monolayer that contains Ni-NTA lipid, a lipid whose head group is functionalized by nickel-nitrilotriacetic acid (Ni-NTA). The Ni-NTA lipids recruit the His-tagged proteins to the lipid monolayer, which can then be lifted off with an EM grid covered with a continuous or holey carbon support film and prepared by negative staining or vitrification for subsequent imaging in the electron microscope.

Here, we introduce the Affinity Grid, a pre-fabricated EM grid with a dried, Ni-NTA lipid-containing monolayer, as a further simplification and extension of the monolayer purification technique. Using His-tagged transferrin-transferrin receptor (Tf-TfR) complex, we show that the Affinity Grid produces specimens equivalent to those obtained by monolayer purification. Although Affinity Grids can be incubated with sample for several hours, an incubation time of only a few minutes usually suffices to produce suitable EM specimens. We also establish that Affinity Grids are resistant to most detergents for a time that depends on the detergent concentration, making them useful for isolating membrane proteins. Finally, we use Affinity Grids to isolate ribosomal complexes from an Escherichia coli extract and the water channel aquaporin-9 (AQP9) from a membrane extract of Sf9 insect cells. Since Affinity Grids can be stored under ambient conditions for several months, they can be pre-fabricated and used whenever needed to prepare specimens for single-particle EM - within minutes and with minimum effort.

Section snippets

Characterization of the Affinity Grid

We used the His-tagged Tf-TfR complex as a test specimen to characterize the Affinity Grid as a tool to prepare specimens for single-particle EM. We first wanted to confirm that Affinity Grids produce samples of the same quality as those prepared with our recently introduced monolayer purification technique.¹ We added His-tagged Tf-TfR complex to an Sf9 cell extract (6 mg/ml protein, 60 mM imidazole) to a final concentration of 0.15 μg/ml. A 3 μl aliquot of this mixture was added to a grid

Affinity Grids in comparison to monolayer purification

We have recently introduced monolayer purification as a rapid and simple technique to purify a His-tagged protein or macromolecular complex from cell extract while simultaneously preparing a specimen suitable for single-particle EM.¹ While it is easy to cast a monolayer over a cell extract and to then pick it up with an EM grid, it does require this procedure be performed for every sample, potentially creating a threshold for the use of monolayer purification. We therefore set out to test

Expression of rpl3, AQP9, TfR and production of Tf-TfR complex

Rpl3 was expressed in E. coli, TfR was expressed in 293-T cells, and the Tf-TfR complex was produced as described.¹ AQP9 was expressed in Sf9 cells as described.⁵

Preparation of cell and membrane extracts

Sf9 and E. coli cell extracts were prepared as described.¹ Membrane extract from Sf9 cells was produced by centrifugation of 50 ml of Sf9 cell extract at 100,000g for 30 min at 4 °C. The pellet containing the cell membranes was homogenized in 100 ml of buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl) containing 2% (w/v) octyl-β,d-glucoside

Acknowledgements

This work was supported by National Institutes of Health grant GM62580 (to S. C. Harrison). The molecular EM facility at Harvard Medical School was established with a generous donation from the Giovanni Armenise Harvard Center for Structural Biology. We thank Matthias Wolf for discussions and advice on image processing.

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Journal of Molecular Biology

The Affinity Grid: A Pre-fabricated EM Grid for Monolayer Purification

Abstract

Introduction

Section snippets

Characterization of the Affinity Grid

Affinity Grids in comparison to monolayer purification

Expression of rpl3, AQP9, TfR and production of Tf-TfR complex

Preparation of cell and membrane extracts

Acknowledgements

J. Struct. Biol.

J. Struct. Biol.

J. Mol. Biol.

J. Mol. Biol.

FEBS Lett.

J. Struct. Biol.