An immunocomplex of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis with the monoclonal Fab fragment L104 was reconstructed from electron micrographs of a negatively stained specimen, using the double-carbon-layer technique. The resulting structure enables a clear visualization of the Fab fragments bound to the four copies of the Aa6 subunit and directly confirms a previous localization of the L104 epitope deduced from two-dimensional image processing. Despite a strong flattening effect produced by the negative-staining technique the orientations of the Fab fragments are well characterized. Moreover, the observation of a central hole within the elbow bends of the Fab fragments provides information about the disposition of the Fabs around their main axis.

Animals↗

PubMedDate not supplied

Three-dimensional reconstruction of Androctonus australis hemocyanin labeled with a monoclonal Fab fragment.

An immunocomplex formed by the 4 x 6 meric hemocyanin of the scorpion Androctonus australis with the monoclonal antibody L104 was studied by cryoelectron microscopy and subjected to three-dimensional (3D) reconstruction. The reconstructed particle reflects the structure of the immunocomplex in its hydrated state and is devoid of the flattening that was previously observed with a negatively stained preparation. A 3D fitting of the X-ray data of the Panulirus interruptus hemocyanin and of the Fab R19.9 to the reconstruction volume allowed the first quantitative measurement of the structural parameters of the antigen, and the localization of the epitope at the surface of subunit Aa6. The independent alignment of the Fabs and the hexamers provides a direct verification for the accuracy of the fit and allows the building of the most detailed model of a cheliceratan 4 x 6meric complex and its attached monoclonal antibodies.

Animals↗

PubMedDate not supplied

Proposal for a new distributed database of macromolecular and subcellular structures from different areas of microscopy.

In this work we address the problem of information access that arises in the field of three-dimensional structure determination, by means of image processing, from data obtained by various types of microscopy. A prototype of a distributed database containing three-dimensional structural information is presented. In this database the volume information is linked, if possible, to other sources of catalogued information such as sequence data, atomic coordinates, and bibliographies. The solution we propose is sufficiently general to be applicable to data in other fields of biomedical science.

Computer Communication Networks↗

PubMedDate not supplied

SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields.

The SPIDER system has evolved into a comprehensive tool set for image processing, making use of modern graphics interfacing in the VMS and UNIX environment. SPIDER and WEB handle the complementary tasks of batch processing and visualization of the results. The emphasis of the SPIDER system remains in the area of single particle averaging and reconstruction, although a variety of other application areas have been added. Novel features are a suite of operations relating to the determination, modeling, and correction of the contrast transfer function and the availability of the entire documentation in hypertext format.

Animals↗

PubMedDate not supplied

Transvenous double-balloon occlusion of the persistent ductus arteriosus: an experimental study.

A new method of transvenous catheter closure has been developed for closure of a persistent ductus arteriosus (PDA), especially in infants and children. The device consists of a 5F or 6F triple-lumen catheter which carries a detachable silicone double-balloon at the tip. After expanding the distal positioning balloon, the proximal occlusion balloon is placed in the PDA and filled to the required size with radiopaque silicone. As the silicone cures inside the balloon, a solid plug occludes the PDA after it is detached from the catheter. In 21 beagles a short segment of subclavian artery was interposed between the main pulmonary artery and the descending aorta, simulating a PDA. The procedure was tested on these models and proved to be suitable. Within one month the plug was covered with fibrous tissue at the pulmonary and aortic ends, thus ensuring permanent occlusion. The long-term follow-up extended for up to two years and confirmed the biocompatibility of the silicone plug.

Angioplasty, Balloon↗

PubMedDate not supplied

Three-dimensional reconstruction of cytochrome oxidase vesicle crystals prepared by cholate solubilization.

Cytochrome oxidase vesicle crystals with long-range order have been obtained from cholate-solubilized, highly purified reconstitutively active preparations. These crystals, which are suitable for electron-microscopic structure investigation, show pgg symmetry in the 0 degree projection. Using Fourier reconstruction and modified back-projection methods, a three-dimensional reconstruction has been obtained at a resolution of 25 A. Our structural results are in agreement with the model of Henderson et al. [J. Mol. Biol. 112, 631 (1977)] obtained for their Triton-derived crystals.

Cholic Acid↗

PubMedDate not supplied

The spectral signal-to-noise ratio resolution criterion: computational efficiency and statistical precision.

This note describes a practical improvement in the computational efficiency of the spectral signal-to-noise ratio (SSNR) resolution criterion for correlation-averaged images. The total set of N images is randomly partitioned into ng subsets, each subset is separately averaged, and a reduced form of the SSNR is computed from these average images. In general, larger values of ng achieve lower statistical uncertainty, while smaller values of ng are computationally more expedient. It is shown that, for negatively stained data, a judicious compromise is achieved with 10 less than or equal to ng less than or equal to 20, regardless of how large N may be.

Microscopy, Interference↗

PubMedDate not supplied

Effect of succinylation on images of negatively stained arrays of mitochondrial outer membrane channels.

The voltage-dependent anion-selective channels of the outer membrane of Neurospora mitochondria occur in two-dimensional crystalline arrays. Electron microscopic images of negatively stained arrays have been compared for normal membranes and membranes pretreated with succinic anhydride, which changes the functional characteristics of the channel. Succinic anhydride does not alter the lattice parameters or the long-range order in the arrays. Also, it has no significant effect on correlation averages of channel arrays embedded in uranyl acetate. Thus, functional changes induced in the channel by succinic anhydride are probably not due to large-scale conformational changes. The distribution of the anionic stain phosphotungstate on the mitochondrial channel arrays is significantly altered by succinic anhydride pretreatment. There are loci on the channels of reduced phosphotungstate accumulation following succinylation. Since phosphotungstate selectively stains positively charged amino acids, it is proposed that these loci may represent clusters of functionally important, exposed basic amino acids.

Anions↗

PubMedDate not supplied

Patch averaging of electron images of GP3*I crystals with variable thickness.

The combination of Fourier and correlation averaging techniques with multivariate statistical analysis and classification, a method known as patch averaging, is used to analyze untilted and tilted images of negatively stained GP32*I crystals, which exhibit variable thicknesses in a single crystal. Within a single image, coherent areas of the same apparent thickness can be distinguished from areas of differing thicknesses. Analysis using the phase relationships among symmetry-related reflections from reconstituted images obtained from untilted micrographs confirms the ability of the method to classify these variable thicknesses properly. Furthermore, the phases from some of the reconstituted images obtained from both untilted and tilted micrographs were found to match well with the phases in a previously determined three-dimensional data set of this crystal with pg symmetry along the crystallographic b axis. These results indicate the utility of the patch averaging procedures in the structural determination of protein crystals with different thicknesses.

Crystallization↗

PubMedDate not supplied

Three-dimensional reconstruction of a complex of human alpha 2-macroglobulin with monomaleimido Nanogold (Au1.4nm) embedded in ice.

Cysteine 949 and glutamine 952 are known to be part of the thiol ester site of each of the four subunits of human alpha 2-macroglobulin (alpha 2M). The hydrolysis of this thiol ester bound to methylamine results in the incorporation of the amine and liberation of a free sulfhydryl group that can be specifically labeled. Therefore, a high-resolution marker specific for the sulfhydryl groups, the monomaleimido Nanogold (Au1.4nm) cluster was used to bind this amino acid. After cryoelectron microscopy, a three-dimensional reconstruction of the alpha 2M-Nanogold conjugates (alpha 2M-Au1.4nm) was achieved, revealing the internal location of the thiol ester sites in the transformed alpha 2M molecules. From this study we propose three possible locations for the cysteine 949.

Cysteine↗

PubMedDate not supplied

Fast 3D motif search of EM density maps using a locally normalized cross-correlation function.

Three-dimensional motif search is becoming increasingly important both in the search for molecular signatures within a tomographic reconstruction, at low resolution, and in the search for atomic structures within high-resolution cryo-EM maps of macromolecular complexes. The present work describes the implementation of a fast local correlation algorithm suitable for template matching in the SPIDER environment. Two examples are given, one in each of the areas of application: (i). within a 7.8A single-particle reconstruction of the Escherichia coli ribosome, four proteins and one RNA structure were located with high accuracy; (ii). within a cryo-tomogram of sarcoplasmic reticulum vesicles, ryanodine receptors were located in positions that agreed with expert knowledge.

Algorithms↗

PubMedDate not supplied

Fast automatic particle picking from cryo-electron micrographs using a locally normalized cross-correlation function: a case study.

Recent progress in single-particle reconstruction methods and cryo-EM techniques has led to the determination of macromolecular structures with unprecedented resolution. The number of particles that goes into the reconstruction is a key determinant in achieving high resolution. Interactive manual picking of particles from an electron micrograph is a very time-consuming, tedious, and inefficient process. We have implemented a fast automatic particle picking procedure in the SPIDER environment. The procedure makes use of template matching schemes and employs a recently developed locally normalized correlation algorithm based on Fourier techniques. As a test, we have used this procedure to pick 70S Escherichia coli ribosomes from a cryo-electron micrograph. Different search strategies including use of a circular mask and asymmetric masks for different orientations of the particle have been explored, and their relative efficiencies are discussed. The results indicate that the procedure can be optimally used to pick ribosomes in a fully automatic way within the limit of selecting less than 10% false positives while missing about 15% of true positives.

Algorithms↗