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Australia’s Great Barrier Reef (GBR) is in severe ecological decline with, for example, large reductions in coral cover and dugong populations. The principal causes of the decline are associated with climate change effects and poor water quality. Poor water quality is due to increased loads of fine sediment, nutrients (nitrogen and phosphorus), and pesticides discharged to the GBR from agricultural sources on the GBR catchment. Management of agricultural practices, primarily in the two principal industries, sugarcane cultivation and rangeland beef grazing, has been in place for 9 years but progress has been slow and inadequate to meet water quality targets. Current water quality management plans are unlikely to reverse this situation largely due to reluctance on the part of the Australian Government to increase funding to the required level to achieve the targets, and a refusal to use existing laws to regulate farming. A number of recommendations, many of which have been suggested in previous analyses, are documented which could allow targets to be reached in the required timelines (i.e., by 2025).

The macrophyte swamp stonecrop has invaded a wide range of wetland habitats across Europe. An experiment was conducted within an invaded fen habitat, which tested whether the presence of grazing disturbance affected the relative abundance of swamp stonecrop, and whether any detected effect was suppressive or facilitative. The abundance of swamp stonecrop and co-occurring resident plants was monitored within fenced grazing exclosures and in adjacent unfenced plots. Swamp stonecrop abundance was higher in the unfenced plots compared to the fenced exclosures (t(87) = 28.974, P < 0.001), whereas the abundance of co-occurring plants was higher in the fenced exclosures compared to the unfenced plots (t(87) = 6.264, P < 0.001). These results indicate that the presence of large vertebrates could facilitate a higher abundance of swamp stonecrop in situations where competitive resident plant species were selectively removed by these grazing animals.

Further refinement of the model using maximum likelihood procedures and reevaluation of the native electron density map has shown that crystals of pig pancreatic alpha-amylase, whose structure we reported more than 15 years ago, in fact contain a substantial amount of carbohydrate. The carbohydrate fragments are the products of glycogen digestion carried out as an essential step of the protein's purification procedure. In particular, the substrate-binding cleft contains a limit dextrin of six glucose residues, one of which contains both alpha-(1,4) and alpha-(1,6) linkages to contiguous residues. The disaccharide in the original model, shared between two amylase molecules in the crystal lattice, but also occupying a portion of the substrate-binding cleft, is now seen to be a tetrasaccharide. There are, in addition, several other probable monosaccharide binding sites. Furthermore, we have further reviewed our X-ray diffraction analysis of alpha-amylase complexed with alpha-cyclodextrin. alpha-Amylase binds three cyclodextrin molecules. Glucose residues of two of the rings superimpose upon the limit dextrin and the tetrasaccharide. The limit dextrin superimposes in large part upon linear oligosaccharide inhibitors visualized by other investigators. By comprehensive integration of these complexes we have constructed a model for the binding of polysaccharides having the helical character known to be present in natural substrates such as starch and glycogen.

Bovine pancreatic ribonuclease A (RNase A) was crystallized from a mixture of small molecules containing basic fuchsin, tobramycin and uridine 5'-monophosphate (U5P). Solution of the crystal structure revealed that the enzyme was selectively bound to U5P, with the pyrimidine ring of U5P residing in the pyrimidine-binding site at Thr45. The structure was refined to an R factor of 0.197 and an R(free) of 0.253.

Pig heart citrate synthase was crystallized from a small-molecule cocktail containing cystamine dihydrochloride, aspartame and benzamidine hydrochloride. The structure was refined to an R factor of 0.179 (R(free) = 0.222) using synchrotron data to a resolution of 1.78 A. The model includes the full-length protein, a chloride ion, a sulfate ion, 305 water molecules and an unexpected moiety attached through a disulfide linkage to Cys184, which was modeled as a half-cystamine molecule generated by disulfide exchange with the cystamine in the small-molecule cocktail.

Proteinase K, a subtilisin-like fungal protease, was crystallized from a cocktail of small molecules containing digalacturonic acid (DGA). The crystal structure was determined to 1.32 Å resolution and refined to an R factor of 0.158. The final model contained, beside the protein, two calcium ions, 379 water molecules, a molecule of DGA and a partially occupied HEPES molecule. The DGA molecule has one sugar moiety disposed exactly on a crystallographic twofold axis; the second ring was not observed. The DGA molecule is bound to two protein molecules across the twofold axis through hydrogen-bonding networks involving Ser150 and water molecules. One of the calcium-ion sites has not been reported previously. This study further illustrates the involvement of small molecules in the crystallization of macromolecules through their ability to form intermolecular lattice interactions. © 2009 International Union of Crystallography All rights reserved.

The Gene Ontology (GO) project (http://www.geneontology.org/) provides a set of structured, controlled vocabularies for community use in annotating genes, gene products and sequences (also see http://www.sequenceontology.org/). The ontologies have been extended and refined for several biological areas, and improvements to the structure of the ontologies have been implemented. To improve the quantity and quality of gene product annotations available from its public repository, the GO Consortium has launched a focused effort to provide comprehensive and detailed annotation of orthologous genes across a number of ‘reference’ genomes, including human and several key model organisms. Software developments include two releases of the ontology-editing tool OBO-Edit, and improvements to the AmiGO browser interface.

Three new crystal forms of satellite panicum mosaic virus (SPMV) were grown and their structures solved from X-ray diffraction data using molecular replacement techniques. The crystals were grown under conditions of pH and ionic strength that were appreciably different then those used for the original structure determination. In rhombohedral crystals grown at pH 8.5 and low ionic strength PEG 3350 solutions, Fourier syntheses revealed segments, ten amino acid residues long, of amino-terminal polypeptides not previously seen, as well as masses of electron density within concavities on the interior of the capsid, which appeared in the neighborhoods of icosahedral five- and threefold axes. The densities were compatible with secondary structural domains of RNA, and they included a segment of double helical RNA of about four to five base pairs oriented, at least approximately, along the fivefold axes. The distribution of RNA observed for SPMV appears to be distinctly different than the encapsidated nucleic acid conformation previously suggested for another satellite virus, satellite tobacco mosaic virus. This study further shows that analysis of viruses in crystals grown under different chemical conditions may reveal additional information regarding the structure of encapsidated RNA. © 2006 Elsevier Inc. All rights reserved.

C(H)2-domain-deleted CC49 (HuCC49DeltaCH2), a recombinant humanized antibody that recognizes the TAG-72 antigen expressed on a variety of human carcinomas, is secreted from cultured cells as a mixture of two homodimeric isoforms. Isoform A contains two covalent interchain disulfide bonds at heavy chain positions 239 and 242, while isoform B fails to develop any interchain disulfide bonds but has 239-242 intrachain disulfide bonds instead. Form A is currently in preclinical development as a therapeutic agent for treating colorectal carcinoma, though form B shows equal efficacy. HuCC49DeltaCH2 form B can be crystallized from sodium formate only in the presence of detergents. X-ray diffraction data were collected on a single cryo-cooled crystal grown with Triton X-100 and the structure was solved by molecular replacement. The model has refined to R=0.246 (R(free)=0.297) for 2.8A data. The antibodies pack in the crystal around crystallographic 2-fold axes as tetramers with approximate 222 symmetry. Atomic force microscopy studies show that this tetrameric structure is the crystal building block and also exists free in the mother liquor. The tetramer is composed of two rings, back-to-back, with a thickness of approximately 83A. Each ring is composed of two antibodies with the complementarity-determining regions (CDR) of the two Fabs of one antibody interacting with the CDR regions of the second antibody in a head-to-head fashion. These rings are approximately 167A long and 112A wide. The C(H)3 domain is inverted with respect to the Fabs when compared to the usual orientation found in conventional antibodies. The polypeptides joining the C(H)3 domains to the Fab portions of the antibody are not seen and are almost certainly disordered. The antigen combining site of HuCC49DeltaCH2 is very similar, but not identical, in topology and charge distribution to that of antibody B72.3, which binds a similar epitope on TAG-72. The combining site consists of a deep cleft, heavily lined with aromatic amino acid side-chains but bounded by numerous charged groups.

Desmodium yellow mottle virus is a 28 nm diameter, T=3 icosahedral plant virus of the tymovirus group. Its structure has been solved to a resolution of 2.7 A using X-ray diffraction analysis based on molecular replacement and phase extension methods. The final R value was 0.151 (R(free)=0.159) for 134,454 independent reflections. The folding of the polypeptide backbone is nearly identical with that of turnip yellow mosaic virus, as is the arrangement of subunits in the virus capsid. However, a major difference in the disposition of the amino-terminal ends of the subunits was observed. In turnip yellow mosaic virus, those from the B and C subunits comprising the hexameric capsomeres formed an annulus about the interior of the capsomere, while the corresponding N termini of the pentameric capsomere A subunits were not visible at all in electron density maps. In Desmodium yellow mottle tymovirus, amino termini from the A and B subunits combine to form the annuli, thereby resulting in a much strengthened association between the two types of capsomeres and an, apparently, more stable capsid. The first 13 residues of the C subunit were invisible in electron density maps. Two ordered fragments of single-stranded RNA, seven and two nucleotides in length, were observed. The ordered water structure of the virus particle was delineated and required 95 solvent molecules per protein subunit.

The molecular structure of satellite tobacco mosaic virus (STMV) has been refined to 1.8 A resolution using X-ray diffraction data collected from crystals grown in microgravity. The final R value was 0.179 and R free was 0.184 for 219,086 independent reflections. The final model of the asymmetric unit contained amino acid residues 13 to 159 of a coat protein monomer, 21 nucleotides, a sulfate ion, and 168 water molecules. The nucleotides were visualized as 30 helical segments of nine base-pairs with an additional base stacked at each 3′ end, plus a “free” nucleotide, not belonging to the helical segments, but firmly bound by the protein. Sulfate ions are located exactly on 5-fold axes and each is coordinated by ten asparagine side-chains. Of the 10,080 structural waters, 168 per asymmetric unit, about 20% serve to bridge the macromolecular components at protein-protein and protein-nucleic acid interfaces. Binding of RNA to the protein involves some salt linkages, particularly to the phosphate of the free nucleotide, but the major contribution is from an intricate network of hydrogen bonds. There are numerous water molecules in the RNA-protein interface, many serving as intermediate hydrogen bond bridges. The sugar-phosphate backbone contributes most of the donors and acceptors for the RNA. The helical RNA conformation is nearest that of A form DNA. The central region of a helical segment is most extensively involved in contacts with protein, and exhibits low thermal parameters which increase dramatically toward the ends. The visible RNA represents approximately 59% of the total nucleic acid in the virion and is derived from the single-stranded genome, which has folded upon itself to form helical segments. Linking of the helices and the free nucleotides in a contiguous and efficient manner severely restricts the disposition of the remaining, unseen nucleic acid. Using the remaining nucleotides it is possible to fold the RNA according to motifs that provide a periodic distribution of RNA structural elements compatible with the icosahedrally symmetrical arrangement seen in the crystallographic structure. The intimate relationship between protein and nucleic acid in STMV suggests an assembly pathway based on the cooperative and coordinated co-condensation of RNA with capsid protein dimers.

Background: The interfacial activation of lipases results primarily from conformational changes in the enzymes which expose the active site and provide a hydrophobic surface for interaction with the lipid substrate. Comparison of the crystallization conditions used and the structures observed for a variety of lipases suggests that the enzyme conformation is dependent on solution conditions. Pseudomonas cepacia lipase (PCL) was crystallized in conditions from which the open, active conformation of the enzyme was expected. Its three-dimensional structure was determined independently in three different laboratories and was compared with the previously reported closed conformations of the closely related lipases from Pseudomonas glumae (PGL) and Chromobacterium viscosum (CVL). These structures provide new insights into the function of this commercially important family of lipases. Results: The three independent structures of PCL superimpose with only small differences in the mainchain conformations. As expected, the observed conformation reveals a catalytic site exposed to the solvent. Superposition of PCL with the PGL and CVL structures indicates that the rearrangement from the closed to the open conformation involves three loops. The largest movement involves a 40 residue stretch, within which a helical segment moves to afford access to the catalytic site. A hydrophobic cleft that is presumed to be the lipidbinding site is formed around the active site. Conclusions: The interfacial activation of Pseudomonas lipases involves conformational rearrangements of surface loops and appears to conform to models of activation deduced from the structures of fungal and mammalian lipases. Factors controlling the conformational rearrangement are not understood, but a comparison of crystallization conditions and observed conformation suggests that the conformation of the protein is determined by the solution conditions, perhaps by the dielectric constant.

The structure of turnip yellow mosaic virus (TYMV) has been solved to 3.2 A resolution and an R-value of 18.7%. The structure is consistent with models based on low resolution X-ray and electron microscopy studies, with pentameric and hexameric protein aggregates protruding from the surface and forming deep valleys at the quasi three-fold axes. The N-terminal 26 residues of the A-subunit are disordered, while those of the B- and C-subunits are seen to interact around the interior of the quasi six-fold cluster where they form an annulus. The three histidine residues of each protein subunit are located in the interior and accessible for interaction with the RNA genome. The appearance of the interior surface of the virus capsid, along with buried surface area calculations, suggest that a pentameric unit is lost during decapsidation.

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.

The crystal structure of anti-idiotopic Fab 409.5.3, made against an E2 specific feline infectious peritonitis virus-neutralizing antibody 730.1.4, has been determined in its free from, at 2.9 A resolution by molecular replacement. This antibody, used as an immmunogen, elicits the production of anti-anti-idiotypic antibodies that in turn neutralize the virus. The structure of the uncomplexed Fab was refined using constrained-restrained least squares minimization and simulated annealing in combination with conjugate gradient techniques to a crystallographic R of 0.22 based on 16,482 unique reflections between 20.0 and 2.9 A. The free antiidiotypic Fab shows, when compared to its complexed form, a 5 degrees rotation of its variable light with respect to its variable heavy domain and rearrangement of complementarity determining region loops, which permits optimization of the stereocomplementarity between interacting molecules. This finding supports the induced fit hypothesis for antibody antigen interaction.

Turnip yellow mosaic virus (TYMV) was purified from Chinese cabbage and crystallized in a form that permits high resolution structural analysis using X-ray diffraction. The crystals have a hexagonal bipyramidal morphology and often achieve dimensions of 1.0 × 1.0 × 0.5 mm. The crystals appear to be of hexagonal space group P6222 with a = b = 525 A, c=315 A, but we cannot strictly rule out the possibility that the space group is P622. They appear different than any crystals of TYMV previously reported. There are three T = 3 virus particles in the unit cell, which implies that one quarter of the particle, or 45 protein subunits, comprises the asymmetric unit of the crystal. Native data have been collected using synchrotron radiation to a resolution of 3.2 A. © 1995 Wiley-Liss, Inc.

Three crystal forms of the sweet-tasting protein thaumatin from the African berry Thaumatococcus daniellii have been grown. These include two naturally occurring isoforms, A and B, that differ by a single amino acid, and a recombinant form of isoform B expressed in yeast. The crystals are of space groups C2 with a = 117.7, b = 44.9, c = 38.0 A, and beta = 94.0 degrees, P2(1)2(1)2(1) with a = 44.3, b = 63.7 and c = 72.7 A, and a tetragonal form P4(1)2(1)2 with a = b = 58.6 and c = 151.8 A. The structures of all three crystals have been solved by molecular replacement and subsequently refined to R factors of 0.184 for the monoclinic at 2.6 A, 0.165 for the orthorhombic at 1.75 A, and 0.181 for the tetragonal, also at 1.75 A resolution. No solvent was included in the monoclinic crystal while 123 and 105 water molecules were included in the higher resolution orthorhombic and tetragonal structures, respectively. A bound tartrate molecule was also clearly visible in the tetragonal structure. The r.m.s. deviations between molecular structures in the three crystals range from 0.6 to 0.7 A for Calpha atoms, and 1.1 to 1.3 A for all atoms. This is comparable to the r.m.s. deviation between the three structures and the starting model. Nevertheless, several peptide loops show particularly large variations from the initial model.

The crystal structure of satellite tobacco mosaic virus (STMV) has been solved by a combination of multiple isomorphous replacement and molecular replacement methods and refined at 2.9 A resolution to a conventional R-factor of 0.215. STMV, a T = 1 icosahedral virus, is the smallest whose structure has been determined. The coat protein is an eight-stranded "Swiss roll" beta-barrel with an amino-terminal strand that extends away from the beta-barrel by more than 60 A. This strand is primarily responsible for quaternary interactions within the capsid. The most arresting feature of the virus structure is the intimate association of each capsid protein dimer with a Watson-Crick base-paired segment of RNA double helix on the interior of the virion. The icosahedral 2-fold axis of each dimer pair is coincident with that of the central base-pair of each helical RNA segment whose helical axis is along the edge of the icosahedron. The helical RNA segments are seven base-pairs in length with a stacked base at each 3' end so that a total of 16 nucleotides is clearly visible. The character of the RNA helix is somewhat different than any of the canonical forms. Assuming full occupancy, then approximately 45% of the total RNA genome is present in the electron density map. The close association of capsid with highly structured nucleic acid suggests that assembly of STMV is likely to be a highly co-operative process involving both protein and RNA. The nucleic acid is distributed within the virion with a high degree of order. The capsid protein is a true double helical RNA binding protein and a number of prominent interactions between protein and RNA can be clearly seen.

Crystal structures of Fab antibody fragments determined by X-ray diffraction characteristically feature four-domain, beta-barrel arrangements. A human antibody Fc fragment has also been found to have four beta-barrel domains. The structures of a few intact antibodies have been solved: in two myeloma proteins, the flexible hinge regions that connect the Fc to the Fab segments were deleted so the molecules were non-functional, structurally restrained, T-shaped antibodies; a third antibody, Kol, had no hinge residues missing but the Fc region was sufficiently disordered that it was not possible to relate its disposition accurately with respect to the Fab components. Here we report the structure at 3.5 A resolution of an IgG2a antitumour monoclonal antibody which contains an intact hinge region and was solved in a triclinic crystal by molecular replacement using known Fc and Fab fragments. The antibody is asymmetric, reflecting its dynamic character. There are two local, apparently independent, dyads in the molecule. One relates the heavy chains in the Fc, the other relates the constant domains of the Fabs. The variable domains are not related by this 2-fold axis because of the different Fab elbow angles of 159 degrees and 143 degrees. The Fc has assumed an asymmetric, oblique orientation with respect to loosely tethered yet almost collinear Fabs. Our study enables the two antigen-binding segments as well as the Fc portion of a functional molecule to be visualized and illustrates the flexibility of these immune response proteins.

Some information and communication technologies (ICT) that support groups have become tightly engrained in the fabric of organizational life, but others have not been as widely adopted (Orlikowski, 1993). This is true of both businesses and educational institutions. For example, many professors and students regularly interact through e-mail. In contrast, groupware systems are not as widely used. In this article, we present research that uses diffusion of innovation (DOI) theory (Rogers, 1995) as the lens through which to study factors that impact intentions to use a groupware application in a higher education setting. This research investigates the following research question: Are adopters’ perceptions of the characteristics of groupware technology related to their intentions to use the technology? Organizations are increasingly making use of ICT to enable distance learning for their employees and, in some cases, customers (Dick, Case, Ruhlman, Van Slyke, & Winston, 2006). Furthermore, numerous academic institutions are implementing and supporting collaborative technologies to support student learning. For instance, Cordon, Anaya, Gonzalez, and Pinzon (2007) report on implementation of a virtual learning center to support learning of 4,000 students in Spain. Leung and Li (2006) describe efforts to create an e-learning environment in Hong Kong. High student dropout rates and low student satisfaction with e-learning remain major drawbacks in such implementations. Despite the presence of online discussion boards, sometimes students feel that there is little interaction in Web-based learning (Chatterjea, 2004). Avoiding failure in distance learning efforts requires better understanding of e-learners and their perception of ICT-based learning technologies.

A new human prostate adenocarcinoma cell line (DuPro-1) has been established from the athymic nude mouse supported xenograft DU5683. This was accomplished by embedding dispersed xenograft cells in 0.1 by 5.0 cm. spaghetti-like strands of Basement Membrane MATRIGEL [BMM (Collaborative Research, Inc.)], a unique technique facilitating the transition to tissue culture. Now passed over 30 times, the cells display anchorage and serum concentration independent growth with a doubling time of 22 to 24 hours. Cells exhibit pronounced morphological differences when grown on BMM coated culture dishes, assuming a pseudoglandular configuration, in contrast to typical homogeneous monolayer growth on plastic culture dishes. Light and electron microscopy show cohesive sheets of anaplastic epithelial cells, consistent with prostate carcinoma. Karyotypic analysis revealed all human chromosomes, near tetraploidy, 10 to 12 markers, and 3 to 4 X chromosomes, without a Y chromosome. Cells injected s.c. or embedded in BMM and implanted in the subrenal capsule space are equally tumorigenic in male and female athymic mice, suggesting that DuPro-1 cells are hormonally insensitive. Embedding cells in BMM may be useful in developing other tissue culture cell lines from neoplasms difficult to initiate in vitro. DuPro-1 should provide a valuable means to study the biology, immunology, and chemosensitivity of human prostate cancer.

Tomato aspermy virus (TAV) is a member of the T = 3 cucumovirus group, and the chrysanthemum strain (C-TAV) has been crystallized in a form suitable for X-ray structural analysis. The crystals, which grow in 14–17% ethanol at pH 8.5, are of orthorhombic space group I222 with unit cell dimensions of a = 295.1 A, b = 320.5 A, and c = 383.6 A. There are two T = 3 virus particles in the unit cell, which means that they must be centered at 0,0,0 and 1/2, 1/2, 1/2 with icosahedral 222 symmetry elements coincident with crystallographic symmetry operators. The asymmetric unit of the crystals, therefore, contains one quarter of a virus particle, or 45 capsid subunits. Native diffraction data to 4 A resolution have been collected using synchrotron radiation, though data appear to be present beyond that resolution. © 1995 Wiley-Liss, Inc.

This project is necessary to insure that replacement of fish screening devices and fishways meet current NMFS design criteria for the protection of all salmonid life stages. The mission of the fish passage program in Northeast Oregon is to protect and enhance fish populations by assisting private landowners, public landowners, irrigation districts and others by maintaining fish screening devices and fishways. These facilities reduce or eliminate fish loss associated with irrigation withdrawals, and as a result insure fish populations are maintained for enjoyment by present and future generations. Assistance is provided through state and federal programs. This can range from basic technical advice to detailed construction, fabrication and maintenance of screening and passage facilities. John Day screens personnel identified 50 sites for fish screen replacement, and one fish passage project. These sites are located in critical spawning, rearing and migration areas for spring chinook, summer steelhead and bull trout. All projects were designed and implemented to meet current NMFS criteria. It is necessary to have a large number of sites identified due to changes in weather, landowner cooperation and access issues that come up as we try and implement our goal of 21 completed projects.

The Malheur River is a 306-kilometer tributary to the Snake River, which drains 12,950 square kilometers. The Malheur River originates in the Blue Mountains and flows into the Snake River near Ontario, Oregon. The climate of the basin is characterized by hot dry summers, occasionally exceeding 38 C, and cold winters that may drop below -29 C. Average annual precipitation is 30 centimeters in the lower reaches. Wooded areas consist primarily of mixed fir and pine forest in the higher elevations. Sagebrush and grass communities dominate the flora in the lower elevations. Efforts to document salmonid life histories, water quality, and habitat conditions have continued in fiscal year 2002. Bull trout Salvelinus confluentus are considered to be cold water species and are temperature-dependant. Due to the interest of bull trout from various state and Federal agencies, a workgroup was formed to develop project objectives related to bull trout. Table 1 lists individuals that participated in the 2002 work group. This report will reflect work completed during the Bonneville Power Administration contract period starting April 1, 2002, and ending March 31, 2003. All tasks were conducted within this timeframe, and a more detailed timeframe may be referred to in each individual more » report. « less

Satellite tobacco mosaic virus (STMV) can undergo at least two physical transitions that significantly alter its mechanical and structural characteristics. At high pH the 17-nm STMV particles expand radially by about 5 Å to yield particles having diameters of about 18 nm. This pH-induced transition is further promoted by aging of the virions and degradation of the RNA, so that swollen particles ultimately appear even at neutral pH. While the native 17-nm particles crystallize as orthorhombic or monoclinic crystals which diffract to high resolution (1.8 Å), the enlarged 18-nm particles crystallize in a cubic form which diffracts to no better than 5 Å. In the transition, not only do the capsid protein subunits move radially outward, but the helical RNA segments with which they interact do as well. This is noteworthy because it demonstrates that the RNA and the protein shell are capable of coordinated movement, and that neither structure is rigidly defined or independent of the other. Using atomic force microscopy, it can be shown that STMV particles, upon drying, lose their mechanical rigidity and undergo deformation. Virions initially 17 nm in diameter shrink to more uniform final sizes than do 18 nm, initially swollen particles. This transition appears to be irreversible, as the particles do not reassume their former size nor structural rigidity upon rehydration. Evidence is also presented that preparations of native virus and their crystals are naturally somewhat heterogeneous and contain a variety of particles of anomalous size. © 2001 Academic Press.

The structure of canavalin was refined to 2.1 and 2.0 A resolution in cubic and hexagonal crystals of space group P2(1)3 and P6(3), respectively. The threefold molecular symmetry is expressed in the symmetry of both crystals, where each identical subunit is an asymmetric unit. The canavalin subunit consists of two very similar domains, each comprised of a core subdomain having Swiss-roll topology with a loop subdomain that contains helices. The refined canavalin models resolved the discrepancy in amino-acid registers of the secondary-structural elements compared with phaseolin. The presence of strand Z in both domains of canavalin was confirmed and a new helix in the loop between strands A and B of each domain was observed. The models were analyzed in terms of the duplicated vicilin domains. Three strictly conserved residues, two glycines and a proline, were identified. The similarity between entire vicilin molecules is greater than that between separate domains of canavalin and phaseolin. Homology modeling of the sucrose-binding protein (SBP) from soybean showed a plausible trimeric assembly of subunits similar to that of vicilins.

The major extracellular protease from the fungus Pencillium cyclopium was crystallized in the presence of p-phenylmethanesulfonyl fluoride (PMSF) and investigated by X-ray diffraction analysis. It was subsequently cloned and the amino acid sequence deduced from its cDNA. Although the sequence is only 49% identical to that of proteinase K of Tritirachium album, the three-dimensional structures of the two proteases are virtually identical. The model for P. cyclopium protease was refined by simulated annealing to an R of 18% at 1.7 A resolution. The greatest variation from the proteinase K polypeptide is in loop 114-134 and is due to the absence of a disulfide bridge in the P. cyclopium protease that is present in proteinase K. A difference was also observed in the orientation of the histidine in the catalytic triad, though this could be due to the presence of PMSF at the active site. The coordination geometry of the strongly bound calcium in the P. cyclopium protease is octahedral and uses some different protein ligands than does proteinase K. In the protease from P. cyclopium there is no cysteine near the active site, nor is there a second calcium binding site as is found in proteinase K, suggesting that neither is important to catalytic activity.

Satellite panicum mosaic virus (SPMV) has been purified from pearl millet and obtained in a variety of different crystal forms, at least two of which appear suitable for high resolution X-ray diffraction analysis. The first is of cubic space group P4232 with a = b = c = 18·1 Å and two virus particles in the unit cell. The second is of a primitive orthorhombic space group with a = 166·1 Å, b = 266·7 Å and c = 269·1 Å, with four virus particles in the unit cell. While the cubic crystal has as its asymmetric unit one twelfth of the icosahedron, or five capsid protein subunits, the asymmetric unit of the orthorhombic crystals is an entire particle. The cubic crystals diffract to at least 2·8 Å resolution. We have also succeeded in crystallizing, but not yet characterizing the master virus, PMV. © 1994 Academic Press Limited.

The structure of pig pancreatic alpha-amylase has been determined by X-ray diffraction analysis using multiple isomorphous replacement in a crystal of space group P2(1)2(1)2(1) (a = 70.6 A, b = 114.8 A, c = 118.8 A) containing nearly 75% solvent. The structure was refined by simulated annealing and Powell minimization, as monitored by 2Fo-Fc difference Fourier syntheses, to a conventional R of 0.168 at 2.1 A resolution. The final model consists of all 496 amino acid residues, a chloride and a calcium ion, 145 water molecules and an endogenous disaccharide molecule that contiguously links protein molecules related by the 2(1) crystallographic operator along x. The protein is composed of a large domain (amino acid residues 1 to 403) featuring a central alpha ta-barrel of eight parallel strands and connecting helices with a prominent excursion between strand beta 3 and helix alpha 3 (amino acid residues 100 to 168). The final 93 amino acid residues at the carboxyl terminus form a second small domain consisting of a compact Greek key beta-barrel. The domains are tightly associated through hydrophobic interfaces. The beta 3/alpha 3 excursion and portions of the central alpha/beta-barrel provide four protein ligands to the tightly bound Ca ion; three water molecules complete the coordination. The Cl- ion is bound within one end of the alpha/beta-barrel by two arginine residues in a manner suggesting a plausible mechanism for its allosteric activation of the enzyme. A crystalline complex of the pancreatic alpha-amylase with alpha-cyclodextrin, a cyclic substrate analog of six glucose residues, reveals, in difference Fourier maps, three unique binding sites. One of the alpha-cyclodextrin sites is near the center of the long polysaccharide binding cleft that traverses one end of the alpha/beta-barrel, another is at the extreme of this cleft. By symmetry this can also be considered as two half sites located at the extremes of the active site cleft. This latter alpha-cyclodextrin displaces the endogenous disaccharide when it binds and, along with the first sugar ring, delineates the extended starch binding site. The third alpha-cyclodextrin binds at an "accessory site" near the edge of the protein and is quite distant from the polysaccharide binding cleft. Its presence explains the multivalency of alpha-amylase binding to dextrins in solution. The extended active site cleft is formed by large, sweeping, connecting loops at one end of the alpha/beta-barrel. These include three sequence segments that are highly conserved among alpha-amylases.(ABSTRACT TRUNCATED AT 400 WORDS)

A complex between an Fab fragment of an E2 specific feline infectious peritonitis virus neutralizing antibody 730.1.4 and Fab fragment from an anti-idiotypic monoclonal antibody 409.5.3, was crystallized from ammonium sulfate using vapor diffusion methods. The complex crystals diffract to about 2·9 A resolution and are of orthorhombic space group P 21221 with a = 75·2 A, b = 80·6 A and c = 187·6 A. There are two Fab molecules, or one idiotype-anti-idiotype complex, comprising the asymmetric unit. The long 187·6 A c axis suggests that the long axis of the complex might lie along this direction. Although small and radiation-sensitive, crystals are suitable for three dimensional structural analysis.

Satellite tobacco mosaic virus (STMV) is the spherical satellite to an obligatory rod-shaped helper tobacco mosaic virus (TMV), which is required for replication. STMV has 60 protein subunits of M(r) 17,500 on a T = 1 icosahedral capsid containing a single-stranded RNA genome of 1,059 bases. STMV appears similar to another virus, STNV, but is approximately 20 per cent smaller. It shows no amino-acid homology or immunological cross-reactivity with either STNV or its host TMV. Here we report the X-ray crystal structure of STMV, which shows that the coat protein of STMV contains a 'Swiss roll' beta-barrel. An amino-terminal strand extends more than 60A and is primarily responsible for quaternary interactions. Each capsid dimer is associated with a segment of genomic RNA double helix comprising seven base pairs. The dyad of each protein dimer is coincident with that of the central base pair of the associated RNA segment whose helix axis is directed along an icosahedral edge. Protein-nucleic acid interactions are extensive. The RNA helices, which have additional stacked bases at their 3' termini, differ significantly from canonical nucleic acid helical forms.

The cationic isozyme of peroxidase isolated from suspension cultures of peanut cells is a heme-containing and calcium-dependent glycoprotein having four covalently attached oligosaccharide chains. Attempts were made to crystallize the glycoprotein for X-ray diffraction analysis, and these have met with some success. Crystals have now been grown that are suitable for a full three-dimensional structural analysis. The crystals are thin plates and we have shown them to be of the orthorhombic space group P2(1)2(1)2(1) with a = 48.1, b = 97.2, c = 146.2 A. The crystals diffract to beyond 2.8 A resolution, appear to be stable to lengthy X-ray exposure, and contain two molecules of 40,000 daltons each in the asymmetric unit.

The Fab fragment of an antibody, made against an E2-specific feline infectious peritonitis virus neutralizing antibody, has been crystallized in a form suitable for X-ray diffraction analysis from PEG 4000 using vapor diffusion methods. The Fab fragment crystals diffract to about 2·9 Å resolution and are of triclinic space group P1. Unit cell dimensions, by which the reciprocal lattice can be indexed, are a = 57·16 A ̊, b = 70·85 A ̊, c = 75·81 A ̊, α = 85·11{ring operator}, β = 121·28{ring operator} and γ = 116·33{ring operator}. There are two Fab fragments comprising the asymmetric unit of the crystals. The presence of a pseudo-mirror plane in the diffraction pattern suggests the presence of at least an approximate dyad axis relating the two Fab fragments within the asymmetric unit. © 1991.

The neutral lipase from the bacteria Pseudomonas fluorescens, marketed under the trade name LpL-200S, has been crystallized in a form suitable for X-ray diffraction analysis from 35% n-propanol at pH 8·5. The crystals are monoclinic prisms and are of space group C2 with a = 91·00 A ̊, b = 47·17 A ̊, c = 35·21 A ̊ and β = 121·43 °. There is one molecule of the protein as the asymmetric unit of the crystals. The diffraction pattern extends to at least 1·6 Å resolution and the crystals are extremely robust in terms of X-ray exposure. © 1991.

A new cell line, SS78, was established from a primary renal cell carcinoma of a Caucasian male. The tissue was dispersed with collagenase, and viable cells were separated by flotation on a Ficoll-Hypaque gradient. In culture, the SS78 cells retained a distinct epithelial morphology, and no fibroblast like cells were seen. The cultured cells were aneuploid with a modal chromosome number of 80 and had several marker chromosomes. Inoculation of the cultured cells into athymic nude mice caused tumors at the sites of inoculation.

The major extracellular protein of Penicillium cyclopium has been isolated from its culture media and purified by ammonium sulfate fractionation, gel, and ion-exchange chromatography. We show this secreted protein to be endopeptidase. The molecular weight is approximately 32,000, the pI is 5.0, and the pH optimum using a variety of protein and synthetic substrates is around 7.0. Inhibition studies show that the protease is not inhibited by pepstatin nor by p-chloromercuribenzoic acid, indicating, respectively, that it is not an aspartyl protease nor a thiol protease. Complete inhibition is observed, however, with phenylmethanesulfonyl fluoride. Three crystal forms suitable for high resolution x-ray diffraction studies have been obtained from this purified protease with reflections being observed to well beyond 3.0 A resolution. One form having a needle morphology is of the orthorhombic crystal class and has space group P2(1)2(1)2(1). The unit cell dimensions are a = 41.9 A, b = 43.2 A, and c = 111.5 A with 1 molecule of the protease occurring in the asymmetric unit. The second form grown at pH values less than 6.0 has a plate morphology, is of orthorhombic space group P2(1)2(1)2(1), and has unit cell dimensions a = 59.12 A, b = 62.33 A, and c = 70.62 A. The third form is polyhedral in habit, is also of space group P2(1)2(1)2(1), and appears when the pH of the mother liquor is greater than 7.0. The cell dimensions of this crystal form are a = 57.07 A, b = 58.82 A, c = 70.79 A, and again there is 1 molecule/asymmetric unit. Three-dimensional structural analysis by x-ray diffraction is now underway. All crystal forms are somewhat denser than the norm having mass to volume ratios of 1.58, 2.00, and 1.85 A3/dalton, respectively.