title: Assessing two-dimensional crystallization trials of small membrane proteins for structural biology studies by electron crystallography.
abstract: Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions. By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size. While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.
authors.affiliation: School of Biology, Georgia Institute of Technology, USA.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Johnson
authors.forename: Frederik
authors.initials: F
authors.lastname: Rudolph
authors.forename: Tina M
authors.initials: TM
authors.lastname: Dreaden
authors.forename: Gengxiang
authors.initials: G
authors.lastname: Zhao
authors.forename: Bridgette A
authors.initials: BA
authors.lastname: Barry
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Schmidt-Krey
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods; D003460:Crystallization / Q000379:methods*; D003461:Crystallography / Q000379:methods*; D004583:Electrons; D008565:Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D008565:Membrane Proteins