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.
Subject(s)
Cryoelectron Microscopy/methods , Chaperonin 60/metabolism , Cryoelectron Microscopy/instrumentation , Crystallography, X-Ray , Electrons , Escherichia coli/ultrastructure , Image Processing, Computer-Assisted , Models, Molecular , Models, Statistical , Specimen HandlingABSTRACT
A fast method for fitting model electron densities into EM reconstructions is presented. The methodology was inspired by the molecular-replacement technique, adapted to take into account phase information and the symmetry imposed during the EM reconstruction. Calculations are performed in reciprocal space, which enables the selection of large volumes of the EM maps, thus avoiding the bias introduced when defining the boundaries of the target density.
Subject(s)
Image Processing, Computer-Assisted , Microscopy, Electron/methods , Models, Molecular , Molecular Conformation , Sensitivity and SpecificityABSTRACT
A new rotation function in Patterson space is described. An image-seeking function can be defined as a criterion of fit between the observed Patterson map and a suitable vector set extracted from a specially calculated Patterson map of the search model. The behaviour of image-seeking functions has appeared to be heavily dependent on certain relations between some statistical parameters of both maps. A new algorithm, which carries out the crucial step of selecting the appropriate vector set from the search model, has been established. As a consequence of the combination of these two preceding results, a new vector-search rotation function has been proposed and tested.
