Reduction of charging in protein electron cryomicroscopy.
Charging causes a loss of resolution in electron cryomicroscopy with biological specimens prepared without a continuous carbon support film. Thin conductive films were deposited onto catalase crystals prepared across holes using ion-beam sputtering and thermal evaporation and evaluated for the effectiveness of charge reduction. Deposits applied by ion-beam sputtering reduced charging but concurrently resulted in structural damage. Coatings applied by thermal evaporation also reduced charging, and preserved the specimen structure beyond 5 A resolution as judged from electron diffraction patterns and images of glucose-embedded catalase crystals tilted to 45 degrees in the microscope. This study demonstrates for the first time the feasibility of obtaining high-resolution data from unstained, unsupported protein crystals with a conductive surface coating.
National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA.
J
J
Brink
H
H
Gross
P
P
Tittmann
M B
MB
Sherman
W
W
Chiu
D002374:Catalase / Q000648:ultrastructure*; D016616:Cryoultramicrotomy; D004583:Electrons; D005613:Freeze Etching / Q000379:methods; D005947:Glucose / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron / Q000379:methods*
D002374:Catalase; D005947:Glucose