title: Lipid monolayer and sparse matrix screening for growing two-dimensional crystals for electron crystallography: methods and examples.
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.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Yeager
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Dryden
authors.forename: Barbie K
authors.initials: BK
authors.lastname: Ganser-Pornillos
mesh_terms: D036022:Capsid Proteins / Q000737:chemistry; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D003461:Crystallography; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
chemical_list: D036022:Capsid Proteins; D008055:Lipids; D011506:Proteins