Cooling-induced wrinkling of thin crystals of biological macromolecules can be prevented by using molybdenum grids

Abstract

It is an important requirement of high resolution electron diffraction and electron microscopy of thin crystals of biological macromolecules that the specimen be flat (i.e. planar) to one degree or less over distances of one micrometer or more. This high degree of specimen flatness is required in order to collect diffraction patterns and images at high tilt angles and high resolution. Imperfect flatness causes the diffraction spots which are perpendicular to the tilt axis to become streaked or blurred, while the spots parallel to the tilt axis remain sharp. If the specimen wrinkling, or mosaic angular spread, is too severe, the diffraction spots overlap one another to make a continuum, and data retrieval becomes impossible (see Figure 3, for an example). Even before that point is reached, the broadening of the diffraction spots makes it increasingly difficult to obtain accurate background-subtracted diffraction intensities, and the signal-to-noise ratio in the computed Fourier transform of high resolution images is severely decreased. In some preparations of thin protein crystals the stringent requirements for specimen flatness can be met with a reasonable rate of success. In the case of bacteriorhodopsin, prepared as large singlecrystal sheets by detergent-induced fusion of the native purple membrane of Halobacterium halobium, the frequency of success is normally very low.