Biomedical subjects

Salvatore Lanzavecchia

Publications and source records attributed to Salvatore Lanzavecchia.

9 recordsLinked to original sources

PubMed2006

Local refinement: an attempt to correct for shrinkage and distortion in electron tomography.

A critical problem in electron tomography is the deformation of the specimen due to radiation, or "shrinkage," which interferes with image alignment and thereby limits resolution. Here, we describe a general strategy for refining preliminary reconstructions which allows the damage due to the shrinkage of plastic-embedded thin sectioned specimens (50-80 nm) to be corrected. The basic steps of the strategy involve: (a) the partition of the preliminary reconstruction into sub-volumes; (b) the extraction of corresponding sub-areas for each sub-volume from the micrographs of the tilt series; (c) the re-projection of each sub-volume according to the orientation parameters; and (d) the refinement of these parameters by correlating each sub-area to the corresponding computed projection. We tested the strategy by refining chemical synapses reconstructed from series imaged with conical, double and single tilt geometries. The results gathered with local refinement were evaluated by visually inspecting the structure of biological membranes in the maps. In an effort to quantify these improvements, we studied the refined maps using correlation criteria and mapped the corrections applied to the orientation parameters in each sub-volume of the reconstruction. Simulation experiments complemented the data gathered by correlation analysis. Based on these criteria, we concluded that local refinement significantly improves the overall quality of the reconstructions of chemical synapses calculated from series imaged with conical and double tilt geometries.

Image Enhancement↗

PubMed2005

Three-dimensional reconstruction of axonemal outer dynein arms in situ by electron tomography.

We present here for the first time a 3D reconstruction of in situ axonemal outer dynein arms. This reconstruction has been obtained by electron tomography applied to a series of tilted images collected from metal replicas of rapidly frozen, cryofractured, and metal-replicated sperm axonemes of the cecidomid dipteran Monarthropalpus flavus. This peculiar axonemal model consists of several microtubular laminae that proved to be particularly suitable for this type of analysis. These laminae are sufficiently planar to allow the visualization of many dynein molecules within the same fracture face, allowing us to recover a significant number of equivalent objects and to improve the signal-to-noise ratio of the reconstruction by applying advanced averaging protocols. The 3D model we obtained showed the following interesting structural features: First, each dynein arm has two head domains that are almost parallel and are obliquely oriented with respect to the longitudinal axis of microtubules. The two heads are therefore positioned at different distances from the surface of the A-tubule. Second, each head domain consists of a series of globular subdomains that are positioned on the same plane. Third, a stalk domain originates as a conical region from the proximal head and ends with a small globular domain that contacts the B-tubule. Fourth, the stem region comprises several globular subdomains and presents two distinct points of anchorage to the surface of the A-tubule. Finally, and most importantly, contrary to what has been observed in isolated dynein molecules adsorbed to flat surfaces, the stalk and the stem domains are not in the same plane as the head.

Animals↗

PubMed2005

Classification of single-projection reconstructions for cryo-electron microscopy data of icosahedral viruses.

We present a novel strategy for classification of heterogeneous electron microscopy data of icosahedral virus particles. The effectiveness of the procedure, which is based on classification of single-projection reconstructions (SPRs), is first investigated using simulated data. Of several reconstruction approaches examined, best results were obtained with algebraic reconstruction techniques (ART) when providing prior information about the reconstruction in the form of a starting volume. The results presented indicate that SPR-classification is sufficiently sensitive to classify assemblies with differences of only a few percent of the total mass. The usefulness of this procedure is illustrated by application to a heterogeneous cryo-electron microscopy dataset of adenovirus mutant dl313, lacking minor coat protein IX. These data were successfully divided into two distinct classes, in agreement with gel analysis and immuno-electron microscopy results. The classes yielded a wildtype-like reconstruction and a reconstruction representing the polypeptide IX-deficient dl313 virion. As the largest difference between these volumes is found at the location previously assigned to the external portion of minor coat protein polypeptide IIIa, questions arise concerning the current adenovirus model.

Adenoviruses, Human↗

PubMed2004

VIVA (from virus variance), a library to reconstruct icosahedral viruses based on the variance of structural models.

VIVA is a software library that obtains low-resolution models of icosahedral viruses from projections observed at the electron microscope. VIVA works in a fully automatic way without any initial model. This feature eliminates the possibility of bias that could originate from the alignment of the projections to an external preliminary model. VIVA determines the viewing direction of the virus images by computation of sets of single particle reconstruction (SPR) followed by a variance analysis and classification of the 3D models. All structures are reduced in size to speed up computation. This limits the resolution of a VIVA reconstruction. The models obtained can be subsequently refined at best with use of standard libraries. Up today, VIVA has successfully solved the structure of all viruses tested, some of which being considered refractory particles. The VIVA library is written in 'C' language and is devised to run on widespread Linux computers.

Analysis of Variance↗

PubMed2003

Structure of functional single AQP0 channels in phospholipid membranes.

Aquaporin-0 (AQP0) is the most prevalent intrinsic protein in the plasma membrane of lens fiber cells where it functions as a water selective channel and also participates in fiber-fiber adhesion. We report the 3D envelope of purified AQP0 reconstituted with random orientation in phospholipid bilayers as single particles. The envelope was obtained by combining freeze-fracture, shadowing and random conical tilt electron microscopy followed by single particle image processing. Two-dimensional analysis of 2547 untilted images produced eight class averages exhibiting "square" and "octagonal" shapes with a continuum of variation. We reconstructed in 3D five class averages that best described the data set. The reconstructions ("molds") appeared as metal cups exhibiting external and internal surfaces. We used the internal surface of the mold to calculate the "imprints" that represent the AQP0 particles protruding from the hydrophobic core of the phospholipid bilayer. The complete envelope of the channel, formed by joining the square and octagonal imprints, described accurately the size, shape, oligomeric state, orientation, and molecular weight of the AQP0 channel inserted in the phospholipid bilayer. Rigid body docking of the atomic model of the aquaporin-1 (AQP1) tetramer showed that the freeze-fracture envelope accounted for the conserved transmembrane domain (approximately 73% similarity between AQP0 and AQP1) but not for the amino and carboxyl termini. We suggest that the discrepancy might reflect differences in the location of the amino and carboxyl termini in the crystal and in the phospholipid bilayer.

Animals↗

PubMed2003

The variance of icosahedral virus models is a key indicator in the structure determination: a model-free reconstruction of viruses, suitable for refractory particles.

A model-free method to determine the three-dimensional structure of icosahedral viruses is described. The novel strategy is based upon the approximate principle that correct virus structures have high variance as do all other well-detailed structures, even wrong ones. The original projections of individual particles are reduced to a radius of 25 pixels and are used to compute single particle reconstruction models by assigning them 1800 different Euler triads. The variance of the models obtained from all projections is stored in maps and a decimation process is carried out. In a first stage, thresholds are adopted for the variance values, and in a second stage, carried out by correspondence analysis and classification, 30 clusters of models are sorted out. The clusters are refined to yield models contained in boxes of 64(3) voxels. The refined models with highest variance and closest similarity represent the correct solution. Once enlarged, these models can be used to align all available projections in their original scale in a customary projection-matching process. The method has proved successful in determining the structures of poliovirus, of the empty and filled capsids of L-A virus, and of a modified capsid of hepatitis B virus.

Capsid↗

PubMed2002

A trajectory-based algorithm to determine and refine Euler angles of projections in three-dimensional microscopy. Improvements and tests.

An improvement of the trajectory matching algorithm is presented, which is based on the use of the derivative of trajectories and of the projection of experimental sinogram lines in the factor space determined by sinogram lines of projections of a model. The algorithm performance is illustrated by use of different phantom structures, to show the effect of symmetry on trajectory matching. A GroEL complex has also been reconstructed from both cryo-negatively stained and unstained frozen-hydrated samples. The refinement of this structure has been carried out by the trajectory matching algorithm as well as by conventional cross-correlation methods. Slight differences among the two results are discussed. The improved trajectory matching algorithm, based on chi2 distances, runs much faster than correlation analysis and looks satisfactory as for the quality of the reconstructed structures.

Journal Article↗

PubMed2002

Symmetry embedding in the reconstruction of macromolecular assemblies via the discrete Radon transform.

In this paper we discuss the embedding of symmetry information in an algorithm for three-dimensional reconstruction, which is based on the discrete Radon transform. The original algorithm was designed for randomly oriented and in principal asymmetric particles. The expanded version presented here covers all symmetry point groups which can be exhibited by macromolecular protein assemblies. The orientations of all symmetry equivalent projections, based on the orientation of an experimental projection, are obtained using global group operators. Further, an improved interpolation scheme for the recovery of the three-dimensional discrete Radon transform has been designed for greater computational efficiency. The algorithm has been tested on phantom structures as well as on real data, a virus structure possessing icosahedral symmetry.

Algorithms↗

PubMed2002

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. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex.

Aeromonas hydrophila↗