The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 Å by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 ؎ 30 Ca 2؉ ions bound to the capsid and 420 ؎ 25 Mg 2؉ions thought to be in the interior of the capsid. Depletion of both Ca 2؉ and Mg 2؉ ions from RCNMV leads to significant structural changes, including (i) formation of 11-to 13-Å-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca 2؉ and Mg 2؉ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.The Tombusviridae family consists of small, icosahedral plant viruses that are transmitted through the soil and infect their hosts via the root system (34). Given that these viruses must survive harsh environments, it is not surprising that they possess unusually stable and robust capsids. This high degree of virion stability raises the question of how the intracellular environment triggers disassembly or minimally exposes the genome for translation. Structural analyses of several plant viruses have revealed that maintenance of a stable capsid conformation is dependent on the presence of divalent cations bound to the capsid (25,33,36). It has been a longstanding hypothesis that swelling and other conformational changes induced by ion extraction from virions are critical for a productive viral life cycle (13). For Tomato bushy stunt virus (TBSV), the type species of the genus Tombusvirus within the Tombusviridae family, it was hypothesized that Ca 2ϩ ions would be released from viral capsids within infected cells, leading to virion swelling sufficient to expose the viral genome.Crystallographic studies of TBSV (28) revealed a capsid formed by 180 chemically identical capsid protein (CP) subunits in three quasiequivalent conformations (A, B, and C). Each CP subunit is composed of three distinct structural domains, which include the RNA-interacting (R), shell (S), and protruding (P) domains. The conformational differences that distinguish the A, B, and C subunits are localized within the hinge regions between the respective S and P domains. These hinges point either down (in A-B dimers) or up (in C-C dimers). In addition, the loop that ...

The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

The structure of the Carnation Mottle Virus (CMtV) capsid protein has been determined at 3.2 A resolution by the method of molecular replacement. Three-dimensional data were collected from a small number of crystals (sp.g. I23, a = 382.6 A) using the synchrotron radiation with an image plate as detector. The coordinates of Tomato Bushy Stunt Virus (TBSV) were used as a searching model. Refinement of the coordinates of 7,479 non-hydrogen atoms performed by the program XPLOR, has led to an R-factor of 18.3%. It was found that the amino acid chain fold of capsid protein is very similar to that in other icosahedral viruses. However, there are some differences in the contact regions between protein subunits and also the lack of the beta-annulus around the 3-fold icosahedral axes. The structural and biochemical results lead us to consider an alternative assembly pathway.