Two-dimensional crystallization technique for imaging macromolecules, with application to antigen–antibody–complement complexes

Abstract

Two-dimensional crystals are formed from macromolecules bound on the surface of a lipid monolayer. A ligand linked to the lipid orientates the binding, and lateral diffusion of the lipids facilitates crystallization. The crystals are suitable for structural analysis by image processing of electron micrographs. An example is the formation of ordered arrays of antibodies on a monolayer of a lipid hapten, and subsequent decoration of these arrays with the first component of complement. Image processing indicates the arrangement of antibodies and the site of complement binding. This approach should be widely applicable to molecular complexes, such as those in replication, protein synthesis, hormone-receptor interaction and metabolic processes.

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

“…EM on lipid monolayers was carried out as described (34)(35)(36). Droplets (Ϸ15 l) of C2A-C2B (0.1 mg͞ml in Tris-buffered saline; 50 mM Tris⅐HCl͞100 mM NaCl, pH 7.4) were placed in Teflon wells (4 mm diameter, 0.5 mm depth) until the liquid surface bulged out.…”

Two‐dimensional crystallization of rabbit C‐reactive protein on lipid monolayers

“…Two-dimensional crystals of macromolecules are particularly suitable for structure determination by electron crystallographic analysis [26]. A general method of 2D crystallization based on the interactions between soluble proteins and ligand-containing lipid layers has been developed by Kornberg and his co-workers [27,28] and has been applied successfully in many cases [29 38]. In the present paper, we report two forms of 2D crystals of rabbit CRP grown on lipid monolayers at the air/water interface under two different conditions based on the specific binding and non-specific electrostatic adsorption of protein with lipid monolayers.…”

Coherent-Pulse 2D Crystallography Using a Free-Electron Laser X-Ray Source

“…As a result, all molecules, or any other biological complexes arranged in a 2D crystal with identical unit cell structure, will give an enhanced signal in a single shot diffraction pattern from FEL sources. This can be especially important for the membrane proteins that in general do not form 3D crystals, but easily form 2D crystalline structures [11,12]. This approach is particularly attractive as the enhancement of the signal-to-noise ratio due to the periodicity improves as the square root of the number of repeated unit cells [13].…”