title: Cryo-negative staining reduces electron-beam sensitivity of vitrified biological particles.
abstract: Beam damage is the main resolution-limiting factor when biological particles are observed by cryoelectron microscopy in a thin vitrified solution film. Furthermore, the low contrast of the specimen frequently makes observation difficult and limits the possibility of image processing. Cryo-negative staining, in which the particles are vitrified in a thin layer of concentrated ammonium molybdate solution, makes it possible to visualize the particles with a much better signal-to-noise ratio (SNR) while keeping the specimen in a good state of preservation. We have observed the Escherichia coli GroEL chaperonin, prepared in a native vitrified solution and by cryo-negative staining after electron exposure from 1000 to 3000e(-)/nm(2). We have compared the resulting three-dimensional models obtained from these different conditions and have tested their fit with the atomic model of the protein subunit obtained from X-ray crystallography. It is found that, down to 1.5-nm resolution, the particles appear to be faithfully represented in the cryo-negatively stained preparation, but there is an approximately 10-fold increase of SNR compared with the native vitrified preparation. Furthermore, for the same range of irradiation and down to the same resolution, the particles seem unaffected by beam damage, whereas the damage is severe in the native vitrified particles.
authors.affiliation: Centre de Microscopie Electronique, 27 rue du Bugnon, 1005, Lausanne, Switzerland. sacha.decarlo@lau.unil.ch
authors.forename: S
authors.initials: S
authors.lastname: De Carlo
authors.forename: C
authors.initials: C
authors.lastname: El-Bez
authors.forename: C
authors.initials: C
authors.lastname: Alvarez-Rúa
authors.forename: J
authors.initials: J
authors.lastname: Borge
authors.forename: J
authors.initials: J
authors.lastname: Dubochet
mesh_terms: D018834:Chaperonin 60 / Q000378:metabolism; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D018360:Crystallography, X-Ray; D004583:Electrons; D004926:Escherichia coli / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D015233:Models, Statistical; D013048:Specimen Handling
chemical_list: D018834:Chaperonin 60