Biomedical subjects

K H Downing

Publications and source records attributed to K H Downing.

59 records · Page 4Linked to original sources

PubMedDate not supplied

Tubulin conformation in zinc-induced sheets and macrotubes.

The protein tubulin is the main constituent of microtubules. Previous studies have shown that zinc ions induce the formation of crystalline sheets and macrotubes of tubulin. Both crystal types are suitable for structural studies by electron crystallography. However, crystallographic structural analysis of tubulin has been hampered by limited crystal size and quality and the inability to control crystal polymorphism. We can obtain well-ordered crystals which are grown upon prolonged incubations (up to 24 hr). The presence of NaCl delays the degradation of the crystals, and addition of the protease inhibitor pepstatin improves crystal quality. The crystal form (sheet or macrotube) can be controlled with incubation conditions. The size of the crystals can reach up to 2 microns in width for the sheets and up to 0.5 microns in diameter for the macrotubes. Both crystal types can reach several micrometers in length. Comparison of the projection maps of the two crystal structures shows that adjacent protofilaments in the macrotubes are shifted by about 6 A relative to their positions in the sheets. Observable changes of monomer shape appear to allow close interprotofilament contacts to be maintained in both crystal forms. Images of glucose-embedded specimens obtained under these conditions give structural information beyond 4 A resolution. Merging of high- and low-resolution data allows for unambiguous assignment of monomer boundaries to high-resolution features.

Animals↗

PubMedDate not supplied

Studies of the structural organization of a bacterial chemoreceptor by electron microscopy.

We used analysis by electron microscopy to obtain structural information about a transmembrane receptor that mediates chemotaxis in Escherichia coli. Two-dimensional arrays of regularly packed particles of the receptor Trg were obtained by reconstitution of purified, detergent-solubilized protein into lipid bilayers. Preliminary image processing of negatively stained arrays revealed an almost square 8.8 x 8.8-nm unit cell and resolved the particles into four peaks of density around a central depression. In certain conditions, reconstituted, Trg-containing bilayers associated into membrane stacks. The regular spacing of the stacks provided a value of 15 nm for the dimension of the receptor normal to the membrane. Using these dimensions, the estimated occupied volume of the structure would be sufficient to contain four monomers of Trg. This tetramer form may be a dimer of two antiparallel or parallel homodimers. Our analysis indicates that a receptor monomer is approximately 4.4 nm at the widest point and 15 nm long. Given the dimensions of the periplasmic domain of the closely related receptor Tars, determined by X-ray crystallography, and a minimum bilayer thickness of 3 nm, the cytoplasmic domain would be approximately 5.0 by 4.4 nm. Higher resolution analysis should reveal additional information about receptor structure.

Bacterial Proteins↗

PubMedDate not supplied

Preservation of 2-D crystals of tubulin for electron crystallography.

Zinc-induced sheets of tubulin are two-dimensional crystalline polymers that constitute an ideal sample for high resolution studies of tubulin by electron crystallography. We show that these 2-dimensional tubulin crystals can be stabilized by taxol against low-temperature depolymerization and degradation with time, easing the way for the preparation of electron microscopy samples. The preservation of the crystals to high resolution has been tested with different embedding media. While glucose-embedded samples diffract poorly, samples embedded in tannin consistently diffract to a resolution of at least 3.5 A. Even better results are obtained by embedding with a combination of tannin and glucose, which improves the flatness of the crystals and allows the collection of isotropic high-resolution data from tilted specimens.

Animals↗

PubMedDate not supplied

Projection map of tubulin in zinc-induced sheets at 4 A resolution.

Tubulin polymerizes into two-dimensional, crystalline sheets in the presence of zinc ions. These sheets are well suited to structural studies by electron crystallography. We have developed conditions for forming sheets which are large and well ordered enough to provide both electron diffraction and image data to better than 4 A resolution. In projection maps calculated from this data, the alpha and beta monomers can be identified within the protofilaments. These results indicate that we should be able to determine the structure of tubulin in these sheets at atomic resolution.

Animals↗

PubMedDate not supplied

Three-dimensional diffractive imaging for crystalline monolayers with one-dimensional compact support.

The use of a compact support constraint along the beam direction is considered as a solution to the phase problem for diffraction by two-dimensional protein crystals. Specifically we apply the iterative Gerchberg-Saxton-Fienup algorithm to simulated three-dimensional transmission electron diffraction data from monolayer organic crystals. We find that oversampling along the reciprocal-lattice rods (relrods) normal to the monolayer alone does not solve the phase problem in this geometry in general. However, based on simulations for a crystalline protein monolayer (lysozyme), we find that convergence is obtained in three dimensions if phases are supplied from a few high resolution electron microscope images recorded at small tilts to the beam direction. In the absence of noise, amplitude-weighted phase residuals of around 5 degrees, and a cross-correlation coefficient of 0.96 between the true and estimated potential are obtained if phases are included from images at tilts of up to 15 degrees. The performance is almost as good in the presence of noise at a level that is comparable to that commonly observed in electron crystallography of proteins. The method should greatly reduce the time and labor needed for data acquisition and analysis in cryo-electron microscopy of organic thin crystals by avoiding the need to record images at high tilt angles.

Algorithms↗