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 Ni2+ 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.
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Acknowledgments
Work in the Yeager laboratory is supported currently by NIH grants R01 HL48908, R01GM066087, R01 GM084545, and P50 GM082545. Parts of this review were abstracted from (2).
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Yeager, M., Dryden, K.A., Ganser-Pornillos, B.K. (2013). Lipid Monolayer and Sparse Matrix Screening for Growing Two-Dimensional Crystals for Electron Crystallography: Methods and Examples. In: Schmidt-Krey, I., Cheng, Y. (eds) Electron Crystallography of Soluble and Membrane Proteins. Methods in Molecular Biology, vol 955. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-176-9_28
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DOI: https://doi.org/10.1007/978-1-62703-176-9_28
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