Electron crystallography of proteins in membranes

Abstract

Electron crystallography has played a vital role in advancing our understanding of proteins in membranes since the 'fluid mosaic model' was proposed in 1972. It is now an established technique to reveal the structures of proteins in their natural bilayer environment and makes possible the study of biological mechanisms through freeze-trapping of transitional states. Thus, images and diffraction patterns of well-ordered, planar and tubular protein-lipid crystals are yielding atomic models, which tell us how the proteins in situ are designed and carry out their membrane-specific tasks. Recent methodological advances and the inclusion of tomographic and cryo-sectioning techniques are enabling detailed information to be obtained from increasingly smaller and more disordered membrane assemblies, extending the potential of this approach.

Figure 1

Protein-lipid arrays used in determining high resolution structures by electron crystallography: two-dimensional crystals (left) and tubular crystals (right). Two-dimensional crystals may contain thousands of unit cells, allowing extensive averaging to improve the signal-to-noise ratio, but need to be tilted to provide the different views required for a three-dimensional analysis. Tubular crystals contain less unit cells, but the molecules are arranged on the surface lattice with helical symmetry, giving rise to many different views and avoiding the need to tilt.

Figure 2

Portion of a density map of tobacco mosaic virus, and fitted polypeptide chains, determined from images of ice-embedded specimens by the iterative helical real-space reconstruction method (reprinted with permission from [31••]). The density map, at a resolution of about 4.5Å, was calculated from images of only 135 virus particles.

Figure 3

Domain structures of a ligand-gated ion channel (ACh receptor, pdb accession code: 2BG9; left) and an ABC transporter (ModB₂C₂, pdb accession code: 2ONK, adapted from [49]; right). In these proteins, the interface between the ligand binding (upper) and transmembrane domains is involved in coupling mechanically distinct conformational changes. The ligand binding domain acts as a controlling element to switch the configuration of helices in the membrane. The interfacial regions crucial to the coupling are in black. For clarity, only two of five ACh receptor subunits are shown.

Figure 4

Comparison of the KcsA potassium channel (pdb accession code: 1BL8, reprinted with permission from [50]; left) with the transmembrane portion of the ACh receptor (pdb accession code:1OED, adapted from [6]; right), illustrating the importance of precise stereo-chemistry in controlling ion conduction across the membrane. Potassium ions flow readily through the narrow selectivity filter of the KcsA channel, because precisely located carbonyl groups lining its surface coordinate with the ion and substitute ideally for the normally tightly bound water molecules. Despite the much wider diameter, the hydrophobic gate region of the ACh receptor forms an effective permeation barrier to potassium (and other) ions, because the ion has no opportunity to shed its hydration shell and so is, in effect, too large to go through. Space filling representations, with the front subunit removed.