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1.
An accurate determination of the protein content of the asymmetric unit can be made conveniently by cross-linking protein crystals with glutaraldehyde then replacing the mother-liquor with pure water. This technique when applied to crystals of adenovirus hexons suggests that the molecular weight assignments of Cornick et al. (1971) were too small and those of Franklin et al. (1971a,b) too large. Density analysis of native hexon crystals by the method of Colman &; Matthews (1971) gives results which are in excellent agreement with those obtained by the method introduced here. 相似文献
2.
R P Bywater C H Carlisle R B Jackson A L Mackay P A Timmins 《Journal of molecular biology》1975,91(3):293-300
Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are , and . The Laue symmetry group is m P31. 相似文献
3.
Markus Marquart Johann Deisenhofer Robert Huber Walter Palm 《Journal of molecular biology》1980,141(4):369-391
The crystal structures of the intact immunoglobulin G1, (λ) Kol and its Fab2 fragment were crystallographically refined at 3.0 Å and 1.9 Å resolution, respectively. The methods used were real space refinement (RLSP) energy and residual refinement (EREF), phase combination, constrained rigid body refinement (CORELS) and difference and Fourier map inspection. The final R-values are 0.24 and 0.26. These analyses allowed the construction of atomic models of parts not seen in detail in the previous analyses at 5 Å and 3 Å resolution, respectively (Colman et al., 1976; Matsushima et al., 1978): i.e. the hinge segment, the hypervariable segments and their intimate interaction with the hinge segment of a crystallographically related molecule.The hinge segment forms a short poly-l-proline double helix from Cys527 to Cys530 (Eu numbering 226 to 230). The preceding segment forms an open turn of helix. This segment and the segment following the poly-l-proline part, which was found to be flexible in Fc fragment crystals (Deisenhofer et al., 1976) probably allow arm and stem movement of the antibody molecule. The combining site of Kol is compared with the combining site of Fab New (Saul et al., 1978). The narrow cleft formed by the hypervariable loops in Kol is filled with aromatic amino acid side-chains. In the crystal, the hypervariable loops contact the hinge and adjacent segments of a related molecule accompanied by a substantial loss in accessible surface area. This contact is preserved in Kol Fab crystals and presumably occurs in the Kol cryoprecipitate. A comparison of the quaternary structures of intact Kol and Fab New showed, in addition to the large change in elbow angle (Colman et al., 1976), changes in lateral domain association. These are discussed in the context of a possible signal transmission from the combining site to the distal end. An attempt was made to model build the IgG3 hinge segment, which is quadruplicated with respect to IgG1 (Michaelsen et al., 1977), on the basis of the Kol hinge structure. A polyproline double helix appeared to be the most plausible model. The Fc part was found to be disordered in intact Kol crystals (Colman et al., 1976). Refinement has reduced the electron density further in the crystal space, where the Fc parts must be located. Disorder, if static, must be fourfold or more in the crystalline state.Intensity measurements on Kol F(ab′)2 and their comparison with intact Kol crystals provide evidence that the disorder is predominantly of a static nature. 相似文献
4.
Pathogen recognition and signal transduction by the Pto kinase 总被引:2,自引:0,他引:2
Jianmin Zhou Xiaoyan Tang Reid Frederick Gregory Martin 《Journal of plant research》1998,111(2):353-356
In tomato, the disease resistance genePto confers resistance to bacterial speck disease by recognizing the expression of a corresponding avirulence gene,avrPto, in the pathogenPseudomonas syringae pv.tomato (Martinet al. 1993). Similar “gene-for-gene” interactions occur in many plant-pathogen associations (Flor 1971). Such recognition events often lead to the activation in the plant of a variety of defense responses including a rapid induction of localized necrosis at the site of infection (the hypersensitive response, HR), increased expression of defense-related genes, production of antimicrobial compounds, lignin formation, and the oxidative burst (Lambet al. 1989, Mehdy 1994). As a result, the pathogen is contained at the infection site and its growth is inhibited.Pto encodes a serine/threonine protein kinase and belongs to a clustered multigene family. Another member of thePto family calledFen confers no known disease resistance, but mediates a hypersensitive-like reaction in the plant to the insecticide fenthion (Martinet al. 1994). We are interested in a number of fundamental questions concerning the Pto signaling pathways. What is the molecular basis of thePto-avrPto gene-for-gene interaction? What are the components involved in thePto-mediated signal transduction chain? How does thePto kinase activate complex defense responses? This paper summarizes our recent progress towards understanding these questions. 相似文献
5.
Fibers of deoxyhemoglobin S obtained directly from lysed sickled red blood cells have been compared with fibers from chromatographically pure deoxyhemoglobin S solutions of known chemical composition. Electron micrographs of negatively stained specimens reveal that the molecular packing within the fibers remains largely invariant with changes in pH, ionic strength, Mg2+ concentration, 2,3-diphosphoglycerate concentration, temperature or the method of deoxygenation.When solutions of chromatographically pure deoxyhemoglobin S are stirred, the fibers align into well defined fascicles. After several hours of stirring, long needles and twisted ribbons develop and in a relatively short time replace the fascicles in solution. With continued stirring all forms are replaced by small crystals. By use of electron microscopy and low-angle X-ray diffraction we have found these crystals to have cell parameters indistinguishable from those of crystals grown in polyethylene glycol and citrate/phosphate buffer at pH 5 to 6 (Wishner et al., 1975a).Our evidence indicates that crystal formation in stirred solutions of deoxyhemoglobin S is the result of a progressive alignment and fusion of the fibers, and that the molecular arrangement within the fibers is closely related to that within the crystal. The remarkable pH invariance of the molecular packing within the fiber and crystal structures is consistent with the dominance of hydrophobic bonding between molecules. The β6-valine contact observed by Wishner et al. (1975b) is apparently the pathological contact responsible for the polymerization of deoxyhemoglobin S in vivo. On the basis of our observations and knowledge of the crystal structure we propose that the deoxyhemoglobin S fiber consists of eight molecular double strands, four of which run in each direction along the length of the fiber. 相似文献
6.
《Journal of molecular biology》1996,262(2):225-242
The crystal structure of the glutamine-binding protein (GlnBP) fromEscherichia coliin a ligand-free “open” conformational state has been determined by isomorphous replacement methods and refined to anR-value of 21.4% at 2.3 Å resolution. There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry. The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules. The structure has root-mean-square deviations of 0.013 Å from “deal” bond lengths and 1.5° from “ideal” bond angles.The GlnBP molecule has overall dimensions of approximately 60 Å × 40 Å × 35 Å and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel β-strands. The small domain contains three α-helices and four parallel and one antiparallel β-strands. The large domain is similar to the small domain but contains two additional α-helices and three more short antiparallel β-strands. A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed.A model of the “closed form” GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and “open form” GlnBP. This model has been successfully used as a search model in the crystal structure determination of the “closed form” GlnBP-Gln complex by molecular replacement methods. The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 Å. Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein. The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described. 相似文献
7.
H.Hasko Paradies Brigitte Zimmer Günther Werz 《Biochemical and biophysical research communications》1977,74(2):397-404
D-Ribulose-1,5-diphosphate carboxylase from was purified, and the gross dimensions were obtained by means of small-angle X-ray scattering measurements in solution. Dissolved single crystals of this enzyme (called “fraction I protein”) gave the same hydrodynamic parameters as the purified form. The molecular weight was found to be 535,000, and a radius of gyration of Rg = 45.5 Å was determined. The experimental scattering curves revealed a geometrical particle of D-Ribulose-1,5-diphosphate carboxylase with gross dimensions of that of a hollow sphere with outer radius of 56 Å and inner radius of 12 Å. Determinations of the diffusion coefficients lead to the conclusion that the enzyme has a spherical shape of almost uniform density. 相似文献
8.
X-ray crystallographic studies of gramicidin A crystallized from methanol (P21) and ethanol (P212121), and of a Cs+ gramicidin A complex crystallized from methanol (P2221, P21212 or P212121) are reported here. The asymmetric unit consists of two molecules of gramicidin A in the native crystals and four molecules in the cesium complex crystal. Patterson analyses show that gramicidin A in these crystals forms a cylindrical helical channel. In the two types of native gramicidin crystals, the diameter of this channel is about 5 å and its length is about 32 å. Cesium ions are bound inside this channel in crystals of the cesium-gramicidin A complex. The channel in this complex is considerably shorter (26 Å) and wider (6·8Å) than in the native forms. The Patterson maps of these three crystal forms are compatible with either the single-stranded β-helix (Urry, 1971) or the double-stranded parallel or anti-parallel, β-helix (Veatch et al., 1974). 相似文献
9.
The carbohydrate binding site of concanavalin A has been identified in crystals of the concanavalin A-methyl alpha-D-mannopyranoside complex and is 35 A from the iodophenol binding site (K. D. Hardman and C. F. Ainsworth (1973), Biochemistry 12,4442), which has been postulated to be adjacent to the carbohydrate-specific binding site (Edelman et al. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 2580). The crystals are orthorhombic in space group C222(1) and crystal denisty measurements indicate a protein mass of four monomers (molecular weight of 104 000) per asymmetric unit. However, the electron density map contains eight monomers/asymmetric unit, revealing lattice disorder. The electron density map with a nominal resolution of 6 A has been solved using three heavy-atom derivatives and the position and orientation of each monomer established. Atomic coordinates of the native protein which has previously been determined (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103) were transposed into this new space group and the gross conformations of the monomers, dimers, and tetramers were found to be very similar to the previous structure. However, some minor differences were apparent even at this resolution. After crystal growth, the methyl alpha-D-mannopyranoside was replaced by o-iodophenyl beta-D-glucopyranoside or methyl 2-iodoacetimido-2-deoxy-alpha-D-glucopyranoside in separate experiments, and difference electron density maps were calculated. The highest peaks for both iodinated sugar derivatives associated with each monomer agreed within a few angstroms of each other and were found near side chains Tyr-12 and -100 and Asp-16 and -208. This region is 10-14 A from the manganese, in good agreement with nuclear magnetic resonance (NMR) studies in solution (C. F. Brewer et al. (1973), Biochemistry 12, 4448) and with the site predicted from crosslinked 1222 crystal studies (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103). 相似文献
10.
We have obtained single crystals of maltose-binding protein (Mr = 40,500) and d-galactose-binding protein (Mr = 32,000), chemoreceptors for active transport and chemotaxis in Escherichia coli. This brings to a total of five the binding proteins that we have thus far crystallized; they include the l-arabinose-binding protein, the leucine, isoleucine, valine-binding protein from Escherichia coli, and a sulfate-binding protein from Salmonella typhimurium. The crystal structure of the l-arabinose-binding protein has been determined at 2.8 Å resolution (Quiocho et al., 1977a). 相似文献
11.
The structure of glycogen phosphorylase alpha at 2.5 A resolution 总被引:10,自引:0,他引:10
The structure of the glucose-inhibited form of glycogen phosphorylase a has been determined at a resolution of 2.5 Å. With the aid of the primary sequence derived by Titani et al. (1977) for this enzyme, we have constructed an atomic model of the 97,400 molecular weight monomer. A substantial improvement in the electron density map over that reported previously (Fletterick et al., 1976b) was achieved by extension of the data set to 2.5 Å and the inclusion of three additional “heavy-atom” derivatives in the phasing procedure. Main-chain and side-chain electron density are clearly resolved in the map, allowing an unambiguous correlation with the published primary structure. The course of the polypeptide backbone in the C-terminal half of the molecule has been modified at two positions from that reported in the 3.0 Å resolution interpretation.The enzyme is clearly organized into two domains, both with packing topology. The catalytic site lies in a crevice at the interface between the two domains. α-d-Glucose, which stabilizes the inactive (T) conformation in the parent crystal, is bound at this site in the C(6′) chair equatorial conformation within 6 Å of the pyridoxal phosphate coenzyme which is covalently bound through the ?-amino group of lysine 679.The larger, N-terminal domain is differentiated by folding architecture and tertiary contacts into two lobes or subdomains which share the same β-sheet backbone: the predominantly helical glycogen storage (maltoheptaose binding) lobe and the N-terminal subdomain. The latter is involved in a variety of protein-protein interactions with the monomer related by the 2-fold axis of the physiological dimer, and contains the serine 14-phosphate moiety and the AMP (positive effector) binding site. The core of the second domain is the complex (βαβ)′ folding unit previously characterized as the nucleotide binding fold (Rao &; Rossmann, 1973). 相似文献
12.
Pore gradient electrophoresis (PGE) in the presence of sodium dodecyl sulfate (SDS) provides a means for high resolution fractionation of multicomponent protein systems and permits estimation of molecular weights for macromolecules ranging from 103 to 106. We have evaluated the performance of several methods used to construct calibration curves for estimation of molecular weights using SDS-PGE. A linear relationship between the logarithm of molecular weight, log (Mr), and the logarithm of the relative mobility, log (Rl), can be obtained for a 30-fold range of molecular weights. However, this range of linearity depends on the choice of the concentration gradient, the degree of crosslinking of the gel, and on the nature of the underlying relationship between the retardation coefficient, KR, and the molecular weight. An empirical relationship, first introduced by Lambin et al. (1976, Anal. Biochem.74, 567) between log (Mr) and the logarithm of the gel concentration at the position reached by the protein, log (%T), provides better linearity over a wider molecular weight range than does the use of log (Rl). We have compared these relatienships by experimental analysis of 10 standard proteins and by a theoretical analysis of an idealized model system. A computer program has been developed which provides appropriate statistical estimation of the molecular weight for an unknown protein, together with its standard error and 95% confidence limits. A new method has also been developed for analysis of nonlinear calibration curves in terms of molecular weight versus distance migrated, based on a theoretically justifiable, physical-chemical model. This model implies that either the relationship between log (Mr) and log (Rl) or the one between log (Mr) and log (%T) will become nonlinear as the range of molecular weight is extended. We suggest that the use of a nonlinear least-squares curve-fitting procedure provides an optimal method for molecular weight estimation when sufficient data are available. Based on these findings, a general strategy is presented for estimation of molecular weights by polyacrylamide gel electrophoresis. 相似文献
13.
Robert K. Moyzis Jacques Bonnet David W. Li Paul O.P. Tso 《Journal of molecular biology》1981,153(4):841-864
The biochemical and biophysical techniques originally introduced by Davidson et al. (1973) and Graham et al. (1974) for the determination of the general organization and length of repetitive and non-repetitive sequences in eukaryotic DNA have been extended and modified. Improvements in the experimental methods employed in these pioneering works have led to novel interpretations and conclusions about mammalian DNA sequence organization. In what is commonly referred to as an interspersion experiment, the average spacing of repetitive DNA regions is inferred from the length dependence of hydroxyapatite binding of radio-labeled tracer DNAs reassociated with an excess of short 200 nucleotide repetitive sequence driver DNA. Studies on Syrian hamster DNA, using an improved procedure for conducting interspersion experiments, suggest that either a frequent cluster in the distribution of non-repetitive DNA sequence lengths occurs at 7200 (±2000) nucleotides or that repetitive sequences are randomly spaced on a number average basis. In contrast, measurements obtained using the traditional methods suggest that a frequent cluster in the distribution of non-repetitive DNA sequence lengths occurs at approximately 1000 nucleotides. When reassociations were conducted at elevated temperatures, to allow only well-matched repetitive sequences to hybridize, the amount of DNA operationally observed as “repetitive” was reduced. Interspersion experiments conducted with Syrian hamster DNA at a reassociation temperature of 75 °C yielded data similar to those obtained by Manning et al. (1975) for Drosophila melanogaster DNA reassociated at 60 °C. 相似文献
14.
Beta-Amylase [1, 4-alpha-D-glucan maltohydrolase, EC 3.2.1.2] has been purified from defatted soybean meal by fractional precipation with ammonium sulfate, ion-exchange chromatography on CM- and DEAE-Sephadex and gel filtration chromatography on Sephadex G-100. Two different components of beta-amylase were crystallized from ammonium sulfate solutions, and the homogeneity of each preparation was confirmed by sedimentation and disc electrophoretic analyses. Both components of soybean beta-amylase formed large single crystals (trigonal crystal system) from 40--50 per cent saturated ammonium sulfate solution buffered at pH 5.4 on dialyzing concentrated protein solution in the apparatus of Zeppezauer et al. Preliminary X-ray diffraction data gave a hexagonal lattice with unit cell dimensions a=86.1 A and c=144.4 A. The space group corresponds to P3121 or P3221, and one asymmetric unit contains one molecule of beta-amylase, assuming a crystal density of 1.25 g/ml and a molecular weight of the enzyme of 60,000 daltons. In this case, the crystal has a volume of 2.53 A-3 per atomic mass unit, and the percentage of protein in the crystal is about 52. 相似文献
15.
An attempt was made to predict the energy value of mixed pig feeds from chemical composition alone, using multiple linear regression analysis. A total of 24 commercial mixed pig feeds were collected on the Belgian market and analysed for various chemical parameters. The digestibility of the Weende nutrients was also determined.For several energy systems used in the European Community (EC), the most reliable regression is given for one to seven independent variables. It was finally found that Nettoenergie Fett (Schiemann et al., 1971), net energy growth (A. Just, 1975, personal communication), digestible energy (Schiemann et al., 1971), metabolizable energy (Just, 1975), Gesamtnährstoffe and total digestible nutrients were most accurately predicted when the following five variables were entered in the regression equation: crude protein, crude fat and the three nitrogen-free extractives (NFE) fractions: starch, invert sugars and “other NFE” (= NFE — starch — invert sugars). 相似文献
16.
Jean-Michel Claverie 《Archives of biochemistry and biophysics》1980,202(1):160-167
A simple and accurate procedure to determine the diffusion coefficient from Active Enzyme Centrifugation experiments is presented. Using computer-simulated concentration distributions we demonstrate that the procedure, derived by Vinograd for the conventional band sedimentation, is suitable to exploit Active Enzyme Centrifugation experiments when the Cohen's Difference Curves Method is used. This new empirical method avoids all the difficulties of the rigorous method previously proposed by Cohen et al., without any loss of accuracy. Optimal conditions are described which allow the determination of the enzyme diffusion coefficient with a 5% uncertainty. Such an easy determination of the sedimentation and diffusion coefficients by the AEC technique can provide a good and rapid estimation of the active enzyme molecular weight, either with a low amount of material or in very impure preparations. 相似文献
17.
Structure of a bacteriochlorophyll-protein from the green photosynthetic bacterium Chlorobium limicola: crystallographic evidence for a trimer 总被引:2,自引:0,他引:2
Crystals of the bacteriochlorophyll-protein from the green photosynthetic bacterium Chlorobium limicola, previously described by J. M. Olson et al. (1969), have been re-examined by X-ray diffraction. The space group is P63, as reported in the earlier work, but revised cell dimensions, , were obtained, leading to a unit cell volume one third of that reported previously. Correction of this error leads to the conclusion that the bacteriochlorophyll-protein complex must be a trimer consisting of three identical subunits arranged about a crystallographic symmetry axis. Also a new trigonal crystal form of the bacteriochlorophyll-protein has been obtained, and is consistent only with a molecule composed of three identical or near-identical subunits. Models of the molecular packing for both crystal forms are presented.The molecular weight of the bacteriochlorophyll-protein complex, determined from crystal density measurements, is (1.53 ± 0.23) × 105, and the overall molecular dimensions are about 55 Å along the trimer axis, and 83 Å at right angles to this. There are probably seven bacteriochlorophyll molecules in each subunit. 相似文献
18.
Two proteinase inhibitors have been isolated from tubers of potato (Solanum tuberosum). Based on N-terminal amino acid sequence homologies, they are members of the Kunitz family of proteinase inhibitors. Potato Kunitz inhibitor-1 (molecular weight 19,500, isoelectric point 6.9) is a potent inhibitor of the animal pancreatic proteinase trypsin, and its amino terminus has significant homology to a recently characterized cathepsin D Kunitz inhibitor from potato tubers (Mares et al. [1989] FEBS Lett 251:94-98). Potato Kunitz inhibitor-2 (molecular weight 20,500, isoelectric point 8.6) is an inhibitor of the microbial proteinase subtilisin Carlsberg; its amino terminus is almost identical to an abundant 22 kilodalton protein from potato tubers (Suh et al. [1990] Plant Physiol 94:40-45) and has significant homology to other Kunitz-type subtilisin inhibitors from small grains. Both Kunitz inhibitors are abundant proteins of the cortex of potato tubers. 相似文献
19.
Comparison of pig heart aconitase (Kennedy et al., 1972) with yeast (Candida lipolytica) aconitase (Suzuki et al., 1973) reveals similarities in molecular weight and iron content but not in sulphide content. Comparison with the Mildvan & Villafranca (1971) pig heart aconitase preparation reveals differences in iron ligands, specific activity and other properties; these differences possibly arise from protein association as pig heart protein associates under a variety of conditions. The electron spin resonance spectrum, g 4.25, and the low molar relaxivity, 473m−1·s−1, of water H+ suggest the presence of high-spin Fe(III) unco-ordinated to water in the enzyme. The iron chromophore on acid titration at 320nm gives a curve with an inflexion at pH4.2. Ten of 16 expected thiol equivalents are titrated with p-hydroxymercuribenzoate suggesting the presence of cystine as well as cysteine residues. Inhibition of the activation of inactive (activatable) enzyme is sigmoidally related to the molar ratio, p-hydroxymercuribenzoate/enzyme with 10–11mol of mercurial compound causing complete inhibition. Active enzyme, free from activating reagents, requires high molar ratios of mercurial compound for rapid inhibition. In terms of p-hydroxymercuribenzoate the enzyme then lacks an essential thiol group. 相似文献
20.
《Journal of molecular biology》2023,435(9):168059
Recent progress in cryo-EM research has ignited a revolution in biological macromolecule structure determination. Resolution is an essential parameter for quality assessment of a cryo-EM density map, and it is known that resolution varies in different regions of a map. Currently available methods for local resolution estimation require manual adjustment of parameters and in some cases necessitate acquisition or de novo generation of so-called “half maps”. Here, we developed CryoRes, a deep-learning algorithm to estimate local resolution directly from a single final cryo-EM density map, specifically by learning resolution-aware patterns of density map voxels through supervised training on a large dataset comprising 1,174 experimental cryo-EM density maps. CryoRes significantly outperforms all of the state-of-the-art competing resolution estimation methods, achieving an average RMSE of 2.26 Å for local resolution estimation relative to the currently most reliable FSC-based method blocres, yet requiring only the single final map as input. Further, CryoRes is able to generate a molecular mask for each map, with accuracy 12.12% higher than the masks generated by ResMap. CryoRes is ultra-fast, fully automatic, parameter-free, applicable to cryo-EM subtomogram data, and freely available at https://cryores.zhanglab.net. 相似文献