ABSTRACT
A new member of the family Closteroviridae was detected in Actinidia chinensis grown in Italy, using next generation sequencing of double-stranded RNA. The virus isolate, named Actinidia virus 1 (AcV-1) has a genome of 18,848 nts in length, a structure similar to the unclassified persimmon virus B (PeVB) and contains 12 open reading frames (ORFs) greater than 6 KDa, one carrying two papain-like leader proteases, a methyltransferase, a helicase and an RNA-dependent RNA polymerase domain. Additional ORFs code for homologs of heat shock protein 70, heat shock protein 90 and a coat protein. Curiously, AcV-1 and PeVB genomes code for a thaumatin-like protein, a peculiarity unreported for other viruses. In phylogenetic analyses both viruses group in a distinct clade evolutionarily related to closteroviruses. The final taxonomic position of AcV-1 within the family Closteroviridae is yet to be clarified.
Subject(s)
Actinidia/virology , Closteroviridae/genetics , Closteroviridae/isolation & purification , Viral Proteins/metabolism , Gene Expression Regulation, Viral , Genome, Viral , Italy , Models, Molecular , Phylogeny , Protein Conformation , Viral Proteins/geneticsABSTRACT
Understanding direct salt effects on protein crystal polymorphism is addressed by comparing different crystal forms (triclinic, monoclinic, tetragonal and orthorhombic) for hen, turkey, bob white quail and human lysozymes. Four new structures of hen egg-white lysozyme are reported: crystals grown in the presence of NapTS diffracted to 1.85 A, of NaI to 1.6 A, of NaNO(3) to 1.45 A and of KSCN to 1.63 A. These new structures are compared with previously published structures in order to draw a mapping of the surface of different lysozymes interacting with monovalent anions, such as nitrate, chloride, iodide, bromide and thiocyanate. An analysis of the structural sites of these anions in the various lysozyme structures is presented. This study shows common anion sites whatever the crystal form and the chemical nature of anions, while others seem specific to a given geometry and a particular charge environment induced by the crystal packing.
Subject(s)
Muramidase/chemistry , Amino Acid Sequence , Animals , Anions , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Protein ConformationABSTRACT
Bovine pancreatic trypsin inhibitor (BPTI) crystallizes under acidic pH conditions in the presence of thiocyanate, chloride and sulfate ions, yielding three different polymorphs in P2(1), P6(4)22 and P6(3)22 space groups, respectively. In all three crystal forms, the same decamer is found in the packing (ten BPTI molecules organized through two perpendicular 2-fold and 5-fold axes as a well-defined and compact object) in contrast to the monomeric crystal forms observed at basic pH conditions. The crystallization of BPTI under acidic conditions (pH 4.5) was investigated by small angle X-ray scattering with both under- and supersaturated BPTI solutions. Data showed the oligomerization of BPTI molecules under all investigated conditions. Accordingly, various mixtures of discrete oligomers (n=1 to 10) were considered. Calculated scattering curves were obtained using models based on the crystallographic structures, and the experimental patterns were analyzed as a linear combination of the model curves using a non-linear curve fitting procedure. The results, confirmed by gel filtration experiments, unambiguously demonstrate the co-existence of two different BPTI particles in solution: a monomer and a decamer, with no evidence of any other intermediates. Moreover, using both approaches, the fraction of decamers was found to increase with increasing salt concentration, even beyond the solubility curve. We therefore propose that at acidic pH, BPTI crystallizes following a two step process: decamers are first built in under- and supersaturated solutions, upon which crystal growth proceeds by decamer stacking. Indeed, those BPTI crystals should best be described as "BPTI decamer" crystals.
Subject(s)
Acids/metabolism , Aprotinin/chemistry , Aprotinin/metabolism , Protein Structure, Quaternary , Amino Acid Sequence , Animals , Anions/metabolism , Binding Sites , Cattle , Chromatography, Gel , Crystallization , Crystallography, X-Ray , Dimerization , Hydrogen-Ion Concentration , Least-Squares Analysis , Models, Molecular , Molecular Sequence Data , Osmolar Concentration , Protein Binding , Software , Solutions , ThermodynamicsABSTRACT
The structure of a monoclinic form of bovine pancreatic trypsin inhibitor (BPTI) crystallized from a thiocyanate solution has been determined and refined at 2.7 A resolution. The space group is P21 with a = 71.56, b = 73.83, c = 64.47 A, beta = 93.9 degrees and Z = 20. The ten independent molecules were located by a multi-body molecular-replacement search as developed in the AMoRe program, starting from a single monomer model (PDB code: 6PTI). The molecular arrangement of the subunits is a decamer resulting from the combination of two orthogonal fivefold and twofold non-crystallographic axes. This builds a globular micelle-like particle which minimizes hydrophobic interactions with the solvent. The refinement was conducted with non-crystallographic symmetry constraints up to a final residual of R = 0.20 (Rfree= 0.26). The root-mean-square deviations from ideal geometry were 0.015 A and 1.6 degrees on bond distances and bond angles, respectively. Several sites for thiocyanate ions were analyzed.
