Figure 1. 2D protein crystals. (a) Sheet-like form: negatively stained crystal of human AQP1 water channel in the form of a lipid bilayer sheet covering the entire field and upon which a relatively large non-crystalline vesicle rests near the top of the figure; the 2D lattice in the image shows repeat units or motifs organized in the form of a ‘burlap’ cloth-like pattern. Scale bar 100 nm. (b) Tubular form: negatively stained tubular crystals of nicotinic acetylcholine receptor formed from the incubation of protein-enriched postsynaptic membranes from Torpedo marmorata electric organs. The repeat units are organized in a helical pattern; individual molecules in the figure appear as bright ‘doughnut-shaped’ objects. Scale bar 50 nm. Part (a) reprinted with permission from the American Chemical Society (Mitra et al., 1994). Part (b) reprinted with permission from Academic Press (Unwin, 1998).

Figure 2. 2D crystallization of membrane proteins by reconstitution. (a) Dialysis method: a dialysis tube, clamped closed at both ends, containing a mixture of detergent solubilized protein and lipid, is placed in an exchange reservoir for the removal of the detergent. The average pore size of the dialysis tube is chosen to allow for the free movement of detergent molecules across the dialysis tube membrane, while retaining protein and lipid molecules. The removal of detergent molecules allows the protein and lipid molecules to form 2D crystals. (b) Bio-Beads method: Bio-Beads (shown here as light-coloured spheres at the bottom of the plastic vial) are added to a mixture of detergent-solubilized protein and lipid to adsorb detergent molecules. This process removes detergent molecules from the mixture, allowing for the formation of 2D crystals.

Figure 3. Crystallization at an air–water interface. (a) A lipid monolayer is first established at the air–water interface. Protein molecules, subsequently added to the bulk solution, concentrate at the preformed lipid monolayer due to electrostatic interactions. 2D crystals form upon incubation. (b) In this case, ligand molecules are linked to lipid head groups; protein molecules are concentrated at the lipid monolayer by protein–ligand binding interactions.