A direct method for determination of membrane electron density profiles on an absolute scale

Abstract

THE principal aim in X-ray diffraction studies of biological membranes and lipid bilayers is to determine the electron density profile across the membrane and to interpret this in terms of the distribution of the component molecules. The derivation of such electron density profiles requires a knowledge of both the amplitudes of the X-ray reflections and their corresponding phase angles. Although the relative amplitudes can be easily determined from a diffraction pattern, the phases of the reflections cannot be directly observed. The determination of these phases has proved to be one of the most difficult and contentious aspects of the membrane diffraction field1–9. In addition, because of the difficulty in measuring absolute intensities10,11, the profiles are, almost invariably, calculated on a relative electron density scale. The lack of an absolute scale has often greatly hindered the interpretation of membrane profiles, particularly with regard to the distribution of the protein molecules9,12–16. We describe here a novel method of directly determining both the phases of the X-ray reflections and an absolute electron density scale, and how the method has been successfully tested on lecithin/cholesterol bilayers.