Is Negative Staining Capable of High Resolution?

Abstract

Everyone presently answers “no” to this question, since this methodology for specimen preparation and preservation commonly is believed to be inherently limited to 20-25Å (2.0-2.5nm) resolution. Achievement of resolution levels smaller than this figure have been published only rarely. This report presents experimental evidence from electron diffraction showing that negative staining can preserve periodic protein structure to the level of at least 4Å.A suspension of bovine liver catalase crystals (orthorhombic: a= 69Å, b= 174Å, c= 206 Å [1]) is deposited on a hydrophilic carbon support and negatively stained using an on-grid protocol [2,3]. For low-dose electron diffraction with a JEOL 100CX transmission electron microscope (l00kV), the set-up for routine selected area diffraction is modified to keep the second condenser lens maximally overfocused; by a defocusing of the diffraction pattern, the grid can be surveyed and suitable thin single crystals centered in the diffraction aperture with only negligible irradiation.