High resolution structural analysis of Helicobacter pylori VacA toxin oligomers by cryo-negative staining electron microscopy.

Helicobacter pylori secretes a vacuolating toxin (VacA) that can assemble into water-soluble oligomeric complexes and insert into membranes to form anion-selective channels. Previous studies have described multiple types of oligomeric VacA structures, including single-layered astral arrays, bilayered forms, and two-dimensional crystalline arrays. In the current study, vitrified VacA complexes were examined by cryo-negative staining electron microscopy, views of the different oligomeric structures in multiple orientations were classified and analyzed, and three-dimensional models of the bilayered forms of VacA were constructed with a resolution of about 19 angstroms.

Spline-based image-to-volume registration for three-dimensional electron microscopy.

This paper presents an algorithm based on a continuous framework for a posteriori angular and translational assignment in three-dimensional electron microscopy (3DEM) of single particles. Our algorithm can be used advantageously to refine the assignment of standard quantized-parameter methods by registering the images to a reference 3D particle model. We achieve the registration by employing a gradient-based iterative minimization of a least-squares measure of dissimilarity between an image and a projection of the volume in the Fourier transform (FT) domain.

Spectral signal-to-noise ratio and resolution assessment of 3D reconstructions.

Measuring the quality of three-dimensional (3D) reconstructed biological macromolecules by transmission electron microscopy is still an open problem. In this article, we extend the applicability of the spectral signal-to-noise ratio (SSNR) to the evaluation of 3D volumes reconstructed with any reconstruction algorithm. The basis of the method is to measure the consistency between the data and a corresponding set of reprojections computed for the reconstructed 3D map.

A multiresolution approach to orientation assignment in 3D electron microscopy of single particles.

Three-dimensional (3D) electron microscopy (3DEM) aims at the determination of the spatial distribution of the Coulomb potential of macromolecular complexes. The 3D reconstruction of a macromolecule using single-particle techniques involves thousands of 2D projections. One of the key parameters required to perform such a 3D reconstruction is the orientation of each projection image as well as its in-plane orientation.

Conversion of a transmembrane to a water-soluble protein complex by a single point mutation.

Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane -- that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin.

Cryo-negative staining reduces electron-beam sensitivity of vitrified biological particles.

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.