Surface crystallisation of the plasma membrane H+-ATPase on a carbon support film for electron crystallography

Abstract

Large two-dimensional crystals of H+-ATPase, a 100 kDa integral membrane protein, were grown directly onto the carbon surface of an electron microscope grid. This procedure prevented the fragmentation that is normally observed upon transfer of the crystals from the air-water interface to a continuous carbon support film. Crystals grown by this method measure approximately 5 microm across and have a thickness of approximately 240 A. They are of better quality than the monolayers previously obtained at the air-water interface, yielding structure factors to at least 8 A in-plane resolution by electron image processing. Unlike most other two-dimensional crystals of membrane proteins they do not contain a lipid bilayer, but consist of detergent-protein micelles of H+-ATPase hexamers tightly packed on a trigonal lattice. The crystals belong to the two-sided plane group p321 (a=b=165 A), containing two layers of hexamers related by an in-plane axis of 2-fold symmetry. The protein is in contact with the carbon surface through its large, hydrophilic 70 kDa cytoplasmic portion, yet due to the presence of detergent in the crystallizing buffer, the hydrophobicity of the carbon surface does not appear to affect crystal formation. Surface crystallisation may be a useful method for other proteins which form fragile two-dimensional crystals, in particular if conditions for obtaining three-dimensional crystals are known, but their quality or stability is insufficient for X-ray structure determination.