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Methods Mol Biol. 2013;955:527-37. doi: 10.1007/978-1-62703-176-9_28.

Lipid monolayer and sparse matrix screening for growing two-dimensional crystals for electron crystallography: methods and examples.

Author information

1
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA, USA.

Abstract

Electron microscopy provides an efficient method for rapidly assessing whether a solution of macromolecules is homogeneous and monodisperse. If the macromolecules can be induced to form two-dimensional crystals that are a single layer in thickness, then electron crystallography of frozen-hydrated crystals has the potential of achieving three-dimensional density maps at sub-nanometer or even atomic resolution. Here we describe the lipid monolayer and sparse matrix screening methods for growing two-dimensional crystals and present successful applications to soluble macromolecular complexes: carboxysome shell proteins and HIV CA, respectively. Since it is common to express recombinant proteins with poly-His tags for purification by metal affinity chromatography, the monolayer technique using bulk lipids doped with Ni(2+) lipids has the potential for broad application. Likewise, the sparse matrix method uses screening conditions for three-dimensional crystallization and is therefore of broad applicability.

PMID:
23132079
PMCID:
PMC4127334
DOI:
10.1007/978-1-62703-176-9_28
[Indexed for MEDLINE]
Free PMC Article

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