Light atom derivatives of structure-preserving sugars are unconventional negative stains.
Although glucose and certain other sugars are known to greatly reduce distortion and denaturation of proteins during drying, use of this monosaccharide as an experimental negative stain does not permit imaging of lattice periodicities in test specimens of thin catalase crystals. However, the potassium and sodium salts of several forms of monophosphorylated glucose (200 mM), diphosphorylated glucose, monosulfated glucose, maltose-1-phosphate, and trehalose-6-phosphate, all dry into a glassy layer and scatter transmitted electrons sufficiently to show the 86 A major periods in catalase crystals. Glucose-6-phosphate provides sufficient image contrast at concentrations from 2 mM (=0.067%) to 500 mM (= 16.8%). Underfocusing increases visualization of the periodic lattice, indicating a large contribution of phase contrast to these images. Upon exposure to the electron beam, thicker regions of derivatized saccharides or pure glucose develop bubbling; this redistribution of dried stain largely can be precluded by imaging with low-dose exposures. Power spectra of images of catalase crystals contained within 200 mM disodium glucose-6-phosphate show that periodic information can be recorded to 21 A; some individual features of dipotassium glucose-6-phosphate distribution within the protein lattice have a measured width of around 5 A. The experimental results demonstrate that structure-preserving mono- and di-saccharides also serve successfully as negative stains after they are coupled to light atom scatterers.
Massover
W H
WH
Department of Anatomy and Cell Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103, USA. massover@anatomy2.umdnj.edu
Marsh
P
P
D000818:Animals; D002241:Carbohydrates; D002374:Catalase / Q000648:ultrastructure; D002417:Cattle; D004187:Disaccharides; D005404:Fixatives; D007089:Image Enhancement; D008854:Microscopy, Electron / Q000379:methods*; D009005:Monosaccharides; D011506:Proteins / Q000648:ultrastructure
D002241:Carbohydrates; D004187:Disaccharides; D005404:Fixatives; D009005:Monosaccharides; D011506:Proteins; D002374:Catalase