Search PubMed⌕ Search

Biomedical subjects

M G Rossmann

Publications and source records attributed to M G Rossmann.

At least 73 records · Page 4Linked to original sources
PubMed1996

Structural refinement of the DNA-containing capsid of canine parvovirus using RSRef, a resolution-dependent stereochemically restrained real-space refinement method.

The canine parvovirus structure (CPV) [Tsao, Chapman, Agbandje, Keller, Smith, Wu, Luo, Smith, Rossmann, Compans & Parrish (1991). Science, 251, 1456-1464] has been refined by a real-space refinement procedure [Chapman (1994). Acta Cryst. A51, 69-801. The fit of an atomic model to electron density was optimized while taking into account the resolution limit of the data and the stereochemistry of the structure. The refined model had a reasonable free R factor [Brtinger (1992). Nature (London), 355, 472-4751 of 0.29. The method is particularly fast and convenient when only a small fraction of the crystallographic asymmetric unit needs to be refined, as is the case when there is high non-crystallographic redundancy. Cycles of refinement for virus capsids were completed in about 1/50th of the time required for equivalent reciprocal-space procedures.

Journal Article↗
PubMed1996

Crystal structure of dimeric HIV-1 capsid protein.

X-ray diffraction analysis of a human immunodeficiency virus (HIV-1) capsid (CA) protein shows that each monomer within the dimer consists of seven alpha-helices, five of which are arranged in a coiled coil-like structure. Sequence assignments were made for two of the helices, and tentative connectivity of the remainder of the protein was confirmed by the recent solution structure of a monomeric N-terminal fragment. The C-terminal third of the protein is mostly disordered in the crystal. The longest helices in the coiled coil-like structure are separated by a long, highly antigenic peptide that includes the binding site of an antibody fragment complexed with CA in the crystal. The site of binding of the Fab, the position of the antigenic loop and the site of cleavage between the matrix protein and CA establish the side of the dimer that would be on the exterior of the retroviral core.

Amino Acid Sequence↗
PubMed1995

Structure determination of coxsackievirus B3 to 3.5 A resolution.

The crystal structure of coxsackievirus B3 (CVB3) has been determined to 3.5 A resolution. The icosahedral CVB3 particles crystallize in the monoclinic space group, P2(1), (a = 574.6, b = 302.1, c = 521.6 A, beta = 107.7 degrees ) with two virions in the asymmetric unit giving 120-fold non-crystallographic redundancy. The crystals diffracted to 2.7 A resolution and the X-ray data set was 55% complete to 3.0,4, resolution. Systematically weak reflections and the self-rotation function established pseudo R32 symmetry with each particle sitting on a 32 special position. This constrained the orientation and position of each particle in the monoclinic cell to near face-centered positions and allowed for a total of six possible monoclinic space-group settings. Correct interpretation of the high-resolution (3.0-3.2 A) self-rotation function was instrumental in determining the deviations from R32 orientations of the virus particles in the unit cell. Accurate particle orientations permitted the correct assignment of the crystal space-group setting amongst the six ambiguous possibilities and for the correct determination of particle positions. Real-space electron-density averaging and phase refinement, using human rhinovius 14 (HRV14) as an initial phasing model, have been carried out to 3.5 A resolution. The initial structural model has been built and refined to 3.5 A resolution using X-PLOR.

Journal Article↗
PubMed1995

Laue diffraction studies of human rhinovirus 14 and canine parvovirus.

Laue diffraction data have been collected from monoclinic crystals of canine parvovirus (CPV), and from cubic crystals of human rhinovirus 14 (HRV14) with and without bound antiviral compounds. In optimal conditions one or two images of HRV14 were sufficient to calculate interpretable electron-density maps of the virus complexes at 3.5 A resolution. The crystals of CPV were of lower symmetry and were more easily damaged by radiation, making it difficult to accumulate a significant amount of useful data. Results on HRV14 are compared in studies on four antiviral compounds where data were collected using both monochromatic and Laue diffraction. Two Laue diffraction images of HRV14 with a point mutation were sufficient to determine the change from a leucine to a valine in VP2.

Journal Article↗
PubMed1995

Structural studies on human rhinovirus 14 drug-resistant compensation mutants.

Structures have been determined of three human rhinovirus 14 (HRV14) compensation mutants that have resistance to the antiviral capsid binding compounds WIN 52035 and WIN 52084. In addition, the structure of HRV14 is reported, with a site-directed mutation at residue 1219 in VP1. A spontaneous mutation occurs at the same site in one of the compensation mutants. Some of the mutations are on the viral surface in the canyon and some lie within the hydrophobic binding pocket in VP1 below the ICAM footprint. Those mutant virus strains with mutations on the surface bind better to cells than does wild-type virus. The antiviral compounds bind to the mutant viruses in a manner similar to their binding to wild-type virus. The receptor and WIN compound binding sites overlap, causing competition between receptor attachment and antiviral compound binding. The compensation mutants probably function by shifting the equilibrium in favor of receptor binding. The mutations in the canyon increase the affinity of the virus for the receptor, while the mutations in the pocket probably decrease the affinity of the WIN compounds for the virus by reducing favorable hydrophobic contacts and constricting the pore through which the antiviral compounds are thought to enter the pocket. This is in contrast to the resistant exclusion mutants that block compounds from binding by increasing the bulk of residues within the hydrophobic pocket in VP1.

Antiviral Agents↗
PubMed1995

Phase refinement and extension by means of non-crystallographic symmetry averaging using parallel computers.

Electron-density averaging, fast Fourier synthesis and fast Fourier analysis programs have been adapted for parallel-computing systems. These have been linked to perform iterative phase improvement and extension utilizing non-crystallographic symmetry and solvent flattening. Various strategies for parallel algorithms have been tested on a variety of computers as a function of the number of computer nodes. Some experimental timing results are discussed.

Journal Article↗
PubMed1995

The structure of coxsackievirus B3 at 3.5 A resolution.

BACKGROUND: Group B coxsackieviruses (CVBs) are etiologic agents of a number of human diseases that range in severity from asymptomatic to lethal infections. They are small, single-stranded RNA icosahedral viruses that belong to the enterovirus genus of the picornavirus family. Structural studies were initiated in light of the information available on the cellular receptors for this virus and to assist in the design of antiviral capsid-binding compounds for the CVBs. RESULTS: The structure of coxsackievirus B3 (CVB3) has been solved to a resolution of 3.5 A. The beta-sandwich structure of the viral capsid proteins VP1, VP2 and VP3 is conserved between CVB3 and other picornaviruses. Structural differences between CVB3 and other enteroviruses and rhinoviruses are located primarily on the viral surface. The hydrophobic pocket of the VP1 beta-sandwich is occupied by a pocket factor, modeled as a C16 fatty acid. An additional study has shown that the pocket factor can be displaced by an antiviral compound. Myristate was observed covalently linked to the N terminus of VP4. Density consistent with the presence of ions was observed on the icosahedral threefold and fivefold axes. CONCLUSIONS: The canyon and twofold depression, major surface depressions, are predicted to be the primary and secondary receptor-binding sites on CVB3, respectively. Neutralizing immunogenic sites are predicted to lie on the extreme surfaces of the capsid at sites that lack amino acid sequence conservation among the CVBs. The ions located on the icosahedral threefold and fivefold axes together with the pocket factor may contribute to the pH stability of the coxsackieviruses.

Amino Acid Sequence↗
PubMed1995

Reciprocal-space molecular-replacement averaging.

The molecular-replacement equations, in which electron-density averaging and skew averaging have been unified, were used in reciprocal space to refine and extend the resolution of phased reflections. A procedure has been developed for the treatment of molecular envelopes of general shape. The equations were successfully applied to the reflection data of bacteriophage phiX174 (60-fold redundancy). Truncation of the G diffraction function beyond the first few nodes did not have a significant effect on the quality of the molecular-replacement equations. Reciprocal-space molecular-replacement averaging should prove to be a useful alternative to real-space averaging. Strategies are discussed that are possible only in reciprocal space.

Journal Article↗
PubMed1995

Nucleocapsid and glycoprotein organization in an enveloped virus.

Alphaviruses are a group of icosahedral, positive-strand RNA, enveloped viruses. The membrane bilayer, which surrounds the approximately 400 A diameter nucleocapsid, is penetrated by 80 spikes arranged in a T = 4 lattice. Each spike is a trimer of heterodimers consisting of glycoproteins E1 and E2. Cryoelectron microscopy and image reconstruction of Ross River virus showed that the T = 4 quaternary structure of the nucleocapsid consists of pentamer and hexamer clusters of the capsid protein, but not dimers, as have been observed in several crystallographic studies. The E1-E2 heterodimers form one-to-one associations with the nucleocapsid monomers across the lipid bilayer. Knowledge of the atomic structure of the capsid protein and our reconstruction allows us to identify capsid-protein residues that interact with the RNA, the glycoproteins, and adjacent capsid-proteins.

Freezing↗
PubMed1995

Single-stranded DNA-protein interactions in canine parvovirus.

BACKGROUND: Parvoviruses are small icosahedral single-stranded (ss) DNA viruses which replicate in rapidly proliferating cells, causing a variety of serious and often lethal diseases in mammals, including humans. The structure of canine parvovirus (CPV) showed an 11-nucleotide oligomeric fragment of its genome bound to 60 equivalent binding sites on the inside surface of the capsid. This provides an opportunity to study the conformation of ssDNA, its interactions with protein, and its role in viral assembly. RESULTS: The icosahedrally ordered part of CPV ssDNA has an unusual loop conformation with the bases pointing outwards and the phosphates surrounding metal ions on the inside. The protein interacts with the bases, making 15 putative hydrogen bonds. The DNA electron density indicates preferences for particular base types in parts of the binding site. Statistical analysis of the genome yields approximately 30 regions with sequences similar to that observed in the structure, demonstrating a low level of sequence specificity for binding to capsid protein. CONCLUSIONS: ssDNA can adopt unusual conformations upon association with protein by using phosphoribose backbone rotamers that are found in tRNA, but not in DNA duplexes. The CPV DNA-protein interactions differ from the non-specific backbone interactions seen in some plant and insect viruses. The sequence specificity, albeit low level, of the protein for CPV DNA may contribute both to distinguishing the viral DNA from other nucleic acids and to the DNA packaging process during viral assembly.

Amino Acids↗
PubMed1995

Ab initio phase determination and phase extension using non-crystallographic symmetry.

Non-crystallographic symmetry (NCS) can be used to improve, extend or find ab initio phases to be associated with a set of observed structure amplitudes, resulting in an interpretable electron-density map. The simplest application is merely to improve the accuracy of the phases by cyclically averaging the electron density, Fourier back-transformation of the modified map, and recomputing a new map with the newly found phases. The first sophistication of this procedure is to phase extend, in successive small steps, the currently available phase information to higher resolution, where only observed amplitudes were previously available. A further sophistication is to initiate the phase extension from very low resolution where a simple geometric model, or an electron microscope image, would be consistent with the chosen resolution. A number of recent examples of virus structure determination exist where such ab initio phasing was successful. The ultimate ab initio phase determination would be to extend phases given only an estimate of the F(000) term.

Crystallography, X-Ray↗
PubMed1994

Calcium ion-induced structural changes in bacteriophage phi X174.

Monoclinic P2(1) crystals of the bacteriophage phi X174 have been incubated with calcium ions (Ca2+) and the induced structural conformational changes studied to 3 A resolution with X-ray crystallographic methods. Three different types of Ca2+ binding sites have been located within the asymmetric unit of the virion. Two sets of sites are associated with the F capsid protein. One set of sites associated with the F protein is in a general position near the icosahedral 3-fold axes of the virus, with the main-chain carbonyl oxygen atoms of residues Gly1321, Asp1421, Met1424 and Ser1426, and the side-chains of Gln1004 and Asp1421 as ligands. The other set of sites associated with the F protein is on the icosahedral 3-fold axes, with the symmetry-related main-chain carbonyl oxygen atoms of Ser1001 and the side-chains of Asn1002 as ligands. The bound Ca2+ induce a conformational change of the amino-terminal residues of the F proteins. A third set of sites, consisting of a pair of Ca2+ on the icosahedral 5-fold axes, are associated with the G spike protein and are concurrently liganded by the symmetry-related carbonyl oxygen side-chains of Asp2117. Concomitant with the binding of Ca2+ to the phage is the rotation of the Asp1209 side-chain of the F protein towards some additional electron density that was not observed in the absence of Ca2+. This density is situated in a shallow depression near the icosahedral 2-fold axes of the virus, and has been tentatively interpreted as a bound glucose molecule that is ordered only in the presence of Ca2+. The putative glucose binding site may be related to the attachment of the virus to cell surface lipopolysaccharides in the initial stages of Escherichia coli infection.

Amino Acid Sequence↗
PubMed1994

The structure of human parvovirus B19 at 8 A resolution.

Empty capsids of the human parvovirus B19, self-assembled in a baculovirus expression system, have been crystallized in a cubic space group P2(1)3 with a = 362 A. In spite of extensive purifications, the crystals diffract X-rays to only 8.0 A resolution. Diffraction data were collected using oscillation photography with synchrotron radiation. The orientations of the particles in the unit cell were determined with a self-rotation function and their positions were obtained with an R-factor search using the known homologous canine parvovirus (CPV) structure. The resultant phases were improved by electron density averaging and solvent flattening to include all the terms between 23.0 and 8.0 A resolution. The central eight-stranded antiparallel beta-barrel, common to many viruses, is situated similarly in B19 with respect to the icosahedral symmetry axes to that observed for feline panleukopenia virus (FPV) and CPV. However, the surface structure of B19 is significantly different from the other known parvoviruses. The most striking difference is that B19 lacks the prominent spikes on the threefold icosahedral axes observed in FPV and CPV. This spike region contains residues involved in host recognition and antigenicity for the latter viruses, showing that there are major differences between subgroups of autonomous parvoviruses.

Amino Acid Sequence↗
PubMed1994

The structure of a neutralized virus: canine parvovirus complexed with neutralizing antibody fragment.

BACKGROUND: Members of the Parvovirus genus cause a variety of diseases in mammals, including humans. One of the major defences against viral infection is the presence of neutralizing antibodies that prevent virus particles from infecting target cells. The mechanism of neutralization is not well understood. We therefore studied the structure of canine parvovirus (CPV) complexed with the Fab fragment of a neutralizing antibody, A3B10, using image reconstruction of electron micrographs of vitrified samples, together with the already known structure of CPV from X-ray crystallographic data. RESULTS: The structure of the complex of CPV with Fab A3B10 has been determined to 23 A resolution. The known CPV atomic structure was subtracted from the electron density of the complex, and the difference map was used to fit the atomic coordinates of a known Fab fragment, HyHEL-5. The long axis of each Fab molecule is oriented in a near radial direction, inclined away from the two-fold axes. The viral epitope consists of 14 amino acid residues found in loops 1, 2 and 3 on the capsid surface, which include previously identified escape mutations. CONCLUSIONS: The mode of Fab binding suggests that the A3B10 neutralizing antibody cannot bind bivalently to the capsid across the two-fold axes, consistent with the observation that whole A3B10 antibody readily precipitates CPV. Since Fab A3B10 can also neutralize the virus, mechanisms of neutralization such as interference with cell attachment, cell entry, or uncoating, must be operative.

Amino Acid Sequence↗
PubMed1994

Analysis of the single-stranded DNA bacteriophage phi X174, refined at a resolution of 3.0 A.

The structure of the bacteriophage phi X174 was examined in a 2.7 A resolution map and refined, using 6.0 A to 3.0 A resolution data with F > or = 5 sigma (F). The final R-factor was 20.9% and the root-mean-square deviation from idealized bond lengths was 0.021 A. The Hendrickson-Konnert refinement was restrained by the phases derived from the molecular replacement icosahedral averaging procedure. The mature phage capsid consists of 60 copies of the F protein with 426 amino acids, the G protein with 175 amino acids and the J protein with 37 amino acids, as well as 12 copies of the H protein with 328 amino acids. The entire polypeptide chain of the F and G protein, all but the first N-terminal residue of the J protein, and 178 solvent molecules were included in the refinement calculations. The secondary structural features of the F, G and J proteins and their interactions with each other are described. The majority of the protein-protein interactions are between the icosahedral 5-fold related interfaces of the F and of the G proteins. These pentameric units of the F and G proteins form the 9S and 6S assembly intermediates, respectively. The J protein lacks any secondary structure and acts as a linking arm between the icosahedral 5-fold related F proteins. Water molecules were introduced only after phase extension to 2.7 A resolution had been completed. The F protein is associated with lower "thermal" parameters and exhibits greater water order in its environment than the G and J proteins. The largest thermal parameters occur in residues on the viral surface. The solvent contributes to the interactions between the proteins. There is an interface of solvent molecules between the F and the G pentamers which stabilizes the pentameric G protein spikes in a crater centered at each of the icosahedral 5-fold vertices of the F protein capsid. Sequence alignments of the F, G and J amino acid sequences for the homologous bacteriophages G4, alpha 3, phi K and phi X174 with respect to the phi X174 structure demonstrated the conservation of functionally important residues on the viral surface.

Amino Acid Sequence↗
PubMed1994

Viral cell recognition and entry.

Rhinovirus infection is initiated by the recognition of a specific cell-surface receptor. The major group of rhinovirus serotypes attach to intercellular adhesion molecule-1 (ICAM-1). The attachment process initiates a series of conformational changes resulting in the loss of genomic RNA from the virion. X-ray crystallography and sequence comparisons suggested that a deep crevice or canyon is the site on the virus recognized by the cellular receptor molecule. This has now been verified by electron microscopy of human rhinovirus 14 (HRV14) and HRV16 complexed with a soluble component of ICAM-1. A hydrophobic pocket underneath the canyon is the site of binding of various hydrophobic drug compounds that can inhibit attachment and uncoating. This pocket is also associated with an unidentified, possibly cellular in origin, "pocket factor." The pocket factor binding site overlaps the binding site of the receptor. It is suggested that competition between the pocket factor and receptor regulates the conformational changes required for the initiation of the entry of the genomic RNA into the cell.

Intercellular Adhesion Molecule-1↗