2D Electron Crystallography of Membrane Protein Single-, Double-, and Multi-Layered Ordered Arrays.

The electron cryo-microscopy (cryo-EM) approach of 2D electron crystallography allows for structure determination of two-dimensional (2D) crystals of soluble and membrane proteins, employing identical principles and methods once 2D crystals are obtained. Two-dimensional crystallization trials of membrane proteins can result in multiple outcomes of ordered arrays, which may be suited for either 2D electron crystallography, helical analysis, or MicroED.The membrane protein 2D crystals used for 2D electron crystallography are either single- or double-layered ordered proteoliposome vesicles or sheet-like membranes. We have developed a cryo-EM grid preparation approach, which allows for the analysis of stacked 2D crystals that are neither suitable for MicroED nor for directly applying 2D electron crystallography. This new grid preparation approach, the peel-blot, uses the capillary force generated by submicron filter paper and mechanical means for the separation of stacked 2D crystals into single-layered 2D crystals, for which standard 2D electron crystallography can then be employed. The preparation of 2D crystals, the peel-blot grid preparation, and the structure determination by 2D electron crystallography are described here.

Inducing two-dimensional crystallization of membrane proteins by dialysis for electron crystallography.

Electron crystallography is an electron cryo-microscopy (cryo-EM) method that is particularly suitable for structure-function studies of small membrane proteins, which are crystallized in two-dimensional (2D) arrays for subsequent cryo-EM data collection and image processing. This approach allows for structural analysis of membrane proteins in a close-to-native, phospholipid bilayer environment. The process of growing 2D crystals from purified membrane proteins by dialysis detergent removal is described in this chapter. A short section covers screening for and identifying 2D crystals by transmission electron microscopy, and in the last section, optimization of the purification to obtain crystals of higher quality is discussed.