Stefano Trapani

AMoRe: classical and modern.

An account is given of the latest developments of the AMoRe package.

Structural Insights Into Viral Determinants of Nematode Mediated Grapevine Fanleaf Virus Transmission.

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematodes feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.

Calculation of spherical harmonics and Wigner d functions by FFT. Applications to fast rotational matching in molecular replacement and implementation into AMoRe

The FFT calculation of spherical harmonics, Wigner D matrices and rotation function has been extended to all angular variables in the AMoRe molecular replacement software. The resulting code avoids singularity issues arising from recursive formulas, performs faster and produces results with at least the same accuracy as the original code. The new code aims at permitting accurate and more rapid computations at high angular resolution of the rotation function of large particles. Test calculations on the icosahedral IBDV VP2 subviral particle showed that the new code performs on the average 1.5 times faster than the original code.

Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase

BackgroundHypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is a central enzyme in the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida cannot synthesize purines de novo and use the salvage pathway to synthesize purine bases, making this an attractive target for antiparasitic drug design.ResultsThe glycosomal HGPRT from Leishmania tarentolae in a catalytically active form purified and co-crystallized with a guanosine monophosphate (GMP) in the active site. The dimeric structure of HGPRT has been solved by molecular replacement and refined against data extending to 2.1 Å resolution. The structure reveals the contacts of the active site residues with GMP.ConclusionComparative analysis of the active sites of Leishmania and human HGPRT revealed subtle differences in the position of the ligand and its interaction with the active site residues, which could be responsible for the different reactivities of the enzymes to allopurinol reported in the literature. The solution and analysis of the structure of Leishmania HGPRT may contribute to further investigations leading to a full understanding of this important enzyme family in protozoan parasites.

Macromolecular crystal data phased by negative-stained electron-microscopy reconstructions.

The combination of transmission electron microscopy with X-ray diffraction data is usually limited to relatively large particles. Here, the approach is continued one step further by utilizing negative staining, a technique that is of wider applicability than cryo-electron microscopy, to produce models of medium-size proteins suitable for molecular replacement. The technique was used to solve the crystal structure of the dodecameric type II dehydroquinase enzyme from Candida albicans (approximately 190 kDa) and that of the orthologous Streptomyces coelicolor protein.

The backbone model of the Arabis mosaic virus reveals new insights into functional domains of Nepovirus capsid

Arabis mosaic virus (ArMV) and Grapevine fanleaf virus (GFLV) are two picorna-like viruses from the genus Nepovirus, consisting in a bipartite RNA genome encapsidated into a 30 nm icosahedral viral particle formed by 60 copies of a single capsid protein (CP). They are responsible for a severe degeneration of grapevines that occurs in most vineyards worldwide. Although sharing a high level of sequence identity between their CP, ArMV is transmitted exclusively by the ectoparasitic nematode Xiphinema diversicaudatum whereas GFLV is specifically transmitted by the nematode X. index. The structural determinants involved in the transmission specificity of both viruses map solely to their respective CP. Recently, reverse genetic and crystallographic studies on GFLV revealed that a positively charged pocket in the CP B domain located at the virus surface may be responsible for vector specificity. To go further into delineating the coat protein determinants involved in transmission specificity, we determined the 6.5 Å resolution cryo-electron microscopy structure of ArMV and used homology modeling and flexible fitting approaches to build its pseudo-atomic structure. This study allowed us to resolve ArMV CP architecture and delineate connections between ArMV capsid shell and its RNA. Comparison of ArMV and GFLV CPs reveals structural differences in the B domain pocket, thus strengthening the hypothesis of a key role of this region in the viral transmission specificity and identifies new potential functional domains of Nepovirus capsid.

The concept of resolution in the domain of rotations

The metric of the SO(3) group of rotations can be used to define the angular resolution of a function of rotations. The resolution is related to the degree of the highest representation present in the expansion of the function in terms of Wigner functions. The peculiar non-Euclidean metric of the rotation domain, however, implies that the terms which effectively contribute to the expansion vary through two-dimensional sections of the rotation domain and are within limiting resolution circles in two-dimensional reciprocal sections. This reconciles an economic sampling of the expansion with the acceleration provided by fast Fourier transform (FFT) techniques.

Combining experimental data for structure determination of flexible multimeric macromolecules by molecular replacement

A major effort has been made by the structural biology community to develop user-friendly software for the use of biologists. However, structural projects become more and more challenging and their solution often relies on a combination of information from various sources. Here, it is described how X-ray data, normal-mode analysis (NMA) and electron-microscopy (EM) data can be successfully combined in order to obtain a molecular-replacement (MR) solution for crystal structures containing multimeric molecules. NMA is used to simulate computationally the inherent internal flexibility of the monomer and thus enhance, together with the crystal noncrystallographic symmetry (NCS), the MR capabilities. NCS is also used to obtain a reliable EM reconstruction, which is then employed as a filter to construct oligomers starting from monomers. The feasibility of the direct use of EM reconstructions as a template for MR when the X-ray and EM data resolutions overlap is also discussed.

Fourier-space TEM reconstructions with symmetry adapted functions for all rotational point groups.

A general-purpose and simple expression for the coefficients of symmetry adapted functions referred to conveniently oriented symmetry axes is given for all rotational point groups. The expression involves the computation of reduced Wigner-matrix elements corresponding to an angle specific to each group and has the computational advantage of leading to Fourier-space TEM (transmission electron microscopy) reconstruction procedures involving only real valued unknowns. Using this expression, a protocol for ab initio view and center assignment and reconstruction so far used for icosahedral particles has been tested with experimental data in other point groups.

Time-Resolved Biological and Perturbation Chemical Crystallography: Laue and Monochromatic Developments

Time-resolved macromolecular x-ray crystallography is a new capability for structural analysis driven by continuing improvements in synchrotron x-ray sources, optics, and detectors (image plates and CCDs). Protein crystal Laue data (stationary crystal and polychromatic x-rays) were recorded at SRS Daresbury station 9.5 and ESRF Grenoble beamline 3, and processed with the Daresbury Laue software package. The Laue method allows exposure times set by the synchrotron electron bunch width, e.g. 50 picoseconds. The instruments and methods developments widen opportunities for perturbation chemical crystallography studies too. A temperature dependent phase transition of a liquid crystal nickel-octahexylphthalocyanine is studied with a rapid readout CCD detector. Structure solution by molecular replacement methods with Laue data is reported for orthorhombic lysozyme. By use of tetragonal lysozyme as a test case it is shown that with fine angular intervals, wide total angular coverage of Laue exposures and the deconvolution of multiples, good connectivity of electron density maps can be realized. The monochromatic rotating crystal method offers possibilites of extremely fast rotations which allow a complete data set to be recorded onto a single image--large-angle oscillation technique (LOT). the processed LOT data looks promising. LOT electron density maps are presented.