摘要:

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 Å 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

ISSN:1399-0047    

DOI:10.1107/S0907444995002988

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The crystal structure of coxsackievirus B3 (CVB3) has been determined to 3.5 Å resolution. The icosahedral CVB3 particles crystallize in the monoclinic space group,P21, (a= 574.6,b= 302.1,c= 521.6 Å, β = 107.7°) with two virions in the asymmetric unit giving 120‐fold non‐crystallographic redundancy. The crystals diffracted to 2.7 Å resolution and the X‐ray data set was 55% complete to 3.0,4, resolution. Systematically weak reflections and the self‐rotation function established pseudoR32 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 Å) self‐rotation function was instrumental in determining the deviations fromR32 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 Å resolution. The initial structural model has been built and

ISSN:1399-0047    

DOI:10.1107/S0907444995002253

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The applicability of the molecular‐replacement (MR) method, implemented through theAMoRepackage [Navaza (1994).Acta Cryst. A50, 157–163], is studied at very low resolution (d>20 Å) and for very large molecular complexes. Due to the nature of the low‐resolution data, specific problems appear. In particular, rotation‐function peaks are very broad and translation functions based on Patterson overlap show large spurious peaks. To solve these problems, the translation function is replaced by a search using amplitude correlation and a systematic three‐dimensional angular search is performed around each rotation‐function peak. Furthermore, these functions are applied in different resolution ranges during the same search. The corresponding algorithms are applied to two cases: the tRNAAsp‐synthetase complex (neutron diffraction data) and a ribosome model crystal (calculated data). This new implementation is shown to solve the problem for a variety of search models, ranging from a detailed atomic model to

ISSN:1399-0047    

DOI:10.1107/S0907444995006743

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

A method is proposed for the solution of the phase problem at very low resolution for macromolecules. It generates randomly a very large number of models, each consisting of a few (two to ten) pseudo‐atoms. The corresponding amplitudes are used for selecting a subset of `best' models by choosing those with the highest correlation with experimental values. The phases calculated from these `best' models are analysed by a clusterization procedure leading to a few possible solutions, from which the correct one can be recognized by simple additional criteria. This method has been successfully applied to the neutron diffraction data of the AspRS–tRNAAspcomplex at 50 Å resolution and to data calculated from a model ribosome crystal at 60 Å r

ISSN:1399-0047    

DOI:10.1107/S0907444995005075

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The three‐dimensional structure of α1‐purothionin (α1‐PT), a wheat‐germ protein and a basic lytic toxin, was previously solved by molecular‐replacement methods using an energy‐minimized predicted model and refined to anR‐factor of 21.6% [Teeter, Ma, Rao&Whitlow (1990).Proteins Struct. Funct. Genet.8, 118–1321. Some deficiencies of the model motivated us to revisit the structure and to continue the refinement. Here we report a significantly improved structure refined to anR‐factor of 15.5% with excellent geometry. The refinement of this relatively low resolution structure (∼2.8 Å) is well suited to test the limitations of classical methods of refinement and to address the problem of overfitting, The final structure contains 434 atoms including 330 protein atoms, 70 waters, three acetates, two glycerols, onesec‐butanol and one phosphate. The key solute molecules (acetate ion and phosphate ion) play a crucial role in the lattice formation. Phosphate and glycerol found in the structure may be important for biologi

ISSN:1399-0047    

DOI:10.1107/S0907444995002964

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The crystal structure of β‐purothionin (β‐PT) has been determined at 1.7 Å resolution. β‐PT and previously solved αl‐PT belong to a family of membrane‐active plant toxins homologous to crambin. (β‐PT crystallizes in the same space group as αl‐PT (1422) but with thecaxis 3 Å longer than (αl‐PT. The unit‐cell dimensions of β‐PT crystals area=b= 53.94 andc= 72.75 Å. Two data sets were collected on a multiwire area detector, each withRsymaround 6.0%, and were merged to get a single data set at 1.7 Å, (Rmerge= 9.6%). The X‐ray structure of αl‐PT was used to build a starting model for β‐PT. The β‐PT model was refined using the programPROLSQfrom 10 to 1.7 Å resolution to anR‐factor of 19.8% with very good geometry. The final structure contains 439 atoms including 337 protein atoms, 77 waters, two acetates, two glycerols and one phosphate. The high‐resolution structure of the β‐PT agreed well with that of the lower resolution αl‐PT structure only after the latter was extensively rerefined. Both refinements revealed phosphate and glycerol molecules which are important in lattice formation. The binding of phosphate and glycerol molecules to purothionins (PT) was confirmed by NMR and was implicated in the biological activity of toxins. Modeling of phospholipid binding to PT based on glycerol and phosphate‐binding site could shed light on the lytic toxicity of this protein‐toxin family. Although the structures of (αl‐PT and β‐PT preserve the overall fold of crambin, they exhib

ISSN:1399-0047    

DOI:10.1107/S0907444995002976

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The crystal structure of native salmon pancreatic elastase (SPE) has been solved by molecular‐replacement methods, and refined by conventional conjugate‐gradient methods and simulated‐annealing techniques. The finalRvalue is 17.2% for 21 389 reflections between 8.0 and 1.61 Å, and the corresponding freeRvalue is 23.9%. The overall tertiary structure of SPE is remarkably similar to that of porcine pancreatic elastase I (PPE), to which it shows about 67% sequence identity. The primary structure of SPE is determined from the electron‐density maps, and only about 15 side chains are somewhat uncertain. Interesting differences between SPE and PPE, are one sequence deletion assigned to position 186, the residue 192 at the entrance of the specificity pocket is substituted from a Gln in PPE to Asn in SPE, and one of the calcium ligands is different. Furthermore, electron density is missing in SPE for the last three residues of the C‐terminal helix. A comparison of the present amino‐acid sequence of SPE with other sequences available indicates that SPE belongs to the class 1 pancre

ISSN:1399-0047    

DOI:10.1107/S0907444995004835

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

A full understanding of the sequence specificity ofEcoRI endonuclease requires structural information on complexes where the DNA contains one `incorrect' base pair; historically, these sites are referred to asEcoRI* sites. They are inherently asymmetric, unlike the canonicalEcoRI site, GAATTC, which possesses a twofold axis of rotational symmetry. All previously determined DNA–EcoRI complexes incorporated this symmetry axis into the space group, requiring the design of `new' oligonucleotides to produce an asymmetric unit appropriate to anEcoRI* complex. The incomplete factorial approach of Carter&Carter [Carter&Carter (1979).J. Biol. Chem.254, 12219–12223.] was used to design the DNA sequence. Factors included the location and type ofEcoRI* substitution and the length and AT richness of the sequences on both sides of the RI site. Co‐crystals were obtained with several sequences, including one with TCGTGGACTTCGTG. Diffraction data were collected from one crystal of this complex to 3.2 Å resolution; the unit‐cell parameters area=b= 123.8 andc= 148.9 Å and the space group isP3221. Unit‐cell and space‐group information was also obtained for theEcoRI* sites AAATTC, GGATTC and GAGTTC. These experiments demonstrated the need for a rapid, economical method that would distinguish DNA‐protein co‐crystals from crystals of protein only. This can be readily achieved with a single small crystal and a staining method based on methylene blue and methyl violet, which stain DNA and

ISSN:1399-0047    

DOI:10.1107/S0907444994005251

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The three‐dimensional structure of the extracellular region of a 60 kDa class II major histocompatibility glycoprotein, HLA‐DR1, was determined to 3.3 Å by X‐ray crystallography using three crystal forms, each containing two molecules per asymmetric unit. Phases were initially determined to 4.2 Å using two crystal forms both containing DR1 from human lymphocytes complexed with a mixture of endogenous peptides. One of these crystal forms also contained a 28 kDa superantigen,Staphylococcus aureusenterotoxin B (SEB), bound to each DR1 molecule. Single‐isomorphous replacement phasing followed by iterative two‐ and fourfold non‐crystallographic real‐space averaging between the two crystal forms resulted in 4.2 Å resolution electron‐density maps from which the paths of the polypeptides could be traced. Cryocrystallography and synchrotron radiation were then used to extend the resolution to 3.3 Å for the two lymphocyte‐derived crystal forms and for a third crystal form grown from DR1 produced in insect cells and complexedin vitrowith a specific antigenic peptide. Iterative sixfold non‐crystallographic real‐space averaging resulted in an electron‐ density map into which 340 of 371 residues could be fit unambiguously. Crystal contacts and the existence of a parallel dimer of the DR1 αβ heterodimer

ISSN:1399-0047    

DOI:10.1107/S0907444995002289

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

Like allc‐type lysozymes, those from rainbow trout act as 1,4‐β‐acetyl‐muramidases to destroy bacteria by cleaving the polysaccharide chains of alternatingN‐acetylglucosamine (NAG) andN‐acetylmuramic acid (NAM) units in the cell walls. Lysozymes also hydrolyse chitin, the analogousN‐acetylglucosamine polymer. The rainbow trout enzymes have been shown to be particularly effective in bacterial defence. We have determined the crystal structures of three complexes between rainbow trout lysozyme (RBTL) and the chito‐oligosaccharides (NAG)2, (NAG)3and (NAG)4to resolutions of 1.8, 2.0 and 1.6 Å, respectively. Crystals of these complexes were obtained by co‐crystallization, and intensity data were collected on a FAST area detector system. Refinement and model building gave finalRvalues of 16.6, 15.9 and 16.5% for the di‐, tri‐ and tetrasaccharide complexes, respectively. The results show that the chito‐oligosaccharides bind to sitesA,BandCas previously observed for complexes between the hen egg‐white lysozyme (HEWL) and a variety of saccharides. The NAG ring in siteDis not bound so deeply and is only slightly distorted towards a half‐chair conformation as observed for the equivalent NAM residue in HEWL. From our results, there is reason to question the position and the degree of strain of theDsaccharide and the mode of binding and importance of two saccharides in sitesEandFfor correct orientation of sugarDand effective hydrolysis of a productive substrate–lysozyme complex. Simple model building study from our structures implies a `left‐sided' binding mode of (NAG)6in the lo

ISSN:1399-0047    

DOI:10.1107/S0907444995005105

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY