Biomedical subjects

N Boisset

Publications and source records attributed to N Boisset.

40 records · Page 3Linked to original sources

PubMedDate not supplied

The effect of overabundant projection directions on 3D reconstruction algorithms.

The experimental process of collecting images from macromolecules in an electron microscope is such that it does not allow for prior specification of the angular distribution of the projection images. As a consequence, an uneven distribution of projection directions may occur. Concerns have been raised recently about the behavior of 3D reconstruction algorithms for the case of unevenly distributed projections. It has been illustrated on experimental data that in the case of a heavily uneven distribution of projection directions some algorithms tend to elongate the reconstructed volumes along the overloaded direction so much as to make a quantitative biological analysis impossible. In answer to these concerns we have developed a strategy for quantitative comparison and optimization of 3D reconstruction algorithms. We apply this strategy to quantitatively analyze algebraic reconstruction techniques (ART) with blobs, simultaneous iterative reconstruction techniques (SIRT) with voxels, and weighted backprojection (WBP). We show that the elongation artifacts that had been previously reported can be strongly reduced. With our specific choices for the free parameters of the three algorithms, WBP reconstructions tend to be inferior to those obtained with either SIRT or ART and the results obtained with ART are comparable to those with SIRT, but at a very small fraction of the computational cost of SIRT.

Algorithms↗

PubMedDate not supplied

Lumbricus terrestris hemoglobin--the architecture of linker chains and structural variation of the central toroid.

The extracellular giant hemoglobin from the earthworm Lumbricus terrestris was reconstructed at 14.9-A resolution from cryo-electron microscope images, using a new procedure for estimating parameters of the contrast transfer (CTF) function. In this approach, two important CTF parameters, defocus and amplitude contrast ratio, can be refined iteratively within the framework of 3D projection alignment procedure, using minimization of sign disagreement between theoretical CTF and cross-resolution curves. The 3D cryo-EM map is in overall good agreement with the recent X-ray crystallography map of Royer et al. (2000, Proc. Natl. Acad. Sci. USA 97, 7107-7111), and it reveals the local threefold arrangement of the three linker chains present within each 1/12 of the complex. The 144 globin chains and 36 linker chains within the complex are clearly visible, and the interdigitation of the 12 coiled-coil helical spokes forming the central toroidal piece is confirmed. Based on these findings, two mechanisms of the dodecameric unit assembly are proposed and termed "zigzag" and "pairwise" polymerizations. However, the detection by cryo-EM of 12 additional rod-like bodies within the toroid raises the possibility that the architecture of the toroid is more complex than previously thought or that yet unknown ligands or allosteric effectors for this oxygen carrier are present.

Animals↗

PubMedDate not supplied

Electron microscopy of alpha 2-macroglobulin with a thiol ester bound ligand.

In order to covalently bind the hydrolyzed thiol ester groups of the human alpha 2-macroglobulin (alpha 2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M(r) = 13,000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed alpha 2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of alpha 2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in alpha 2M. They also suggest that the four thiol esters are located near the center of the alpha 2M molecule.

Animals↗

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↗