A geometry force field which converts low-resolution X-ray models to structures with properties found at ultra high resolution

A geometry optimization force field was developed using ultra high-resolution structures and tested using high- and low-resolution X-ray structures. Protein and small molecule X-ray data was used. When applied to ultra high-resolution structures the force field conserves the internal geometry and local strain energy. When applied to low-resolution structures there is a small change in geometry accompanied by a large drop in local strain energy. Although optimization causes only small structural changes in low-resolution X-ray models, it dramatically modifies profiles for hydrogen bonding, Van der Waals contact, bonded geometry, and local strain energy, making them almost indistinguishable from those found at high resolution. Further insight into the effect of the force field was obtained by comparing geometries of homologous proteins before and after geometry optimization. Optimization causes homologous regions of structures to become similar in internal geometry and energies. Once again, the changes only require small atomic movements. These findings provide insights into the structure of molecular complexes. The new force field contains only short-range interatomic potential functions. Its effectiveness shows that local geometries are determined by short-range interactions which are well modeled by the force field. Potential applications of this study include detection of possible structural errors, correction of errors with minimal change in geometry, improved understanding and prediction of the effects of modifying ligands or proteins, and computational addition of structural water.     

Crystals of crotoxin suitable for high resolution x-ray diffraction analysis

The phospholipasic presynaptic neurotoxin, crotoxin, has been crystallized in a morphology suitable for single crystal x-ray diffraction analysis. The conditions for growth and the unit cell parameters (P4(1)22 or P4(3)22, a = b = 38.5 A, c = 256.9 A, 1 molecule/asymmetric unit) are similar to the very thin plate-like crystals which have been studied with electron diffraction and electron microscopy by Chiu and his colleagues (Jeng, T.-W., Chiu, W., Zemlin, F., and Zeitler, E. (1984) J. Mol. Biol. 175, 93 - 97). These two macroscopic crystal morphologies of what is likely to be a very similar, if not identical, lattice structure will permit the complementary application of electron diffraction/microscopy and x-ray diffraction to understanding the structural basis of the interactions between a phospholipasic neurotoxin and its membrane target.     

Two crystal forms of the lentil lectin diffract to high resolution.

The legume lectins are an important class of polysaccharide-binding proteins with a wide range of biochemical and immunological applications. Two high-resolution crystal forms are obtained for the lentil (Lens culinaris) lectin: a monoclinic P21 and an orthorhombic P212121. The unit cell dimensions for the monoclinic form are a = 58.0 A, b = 56.0 A, c = 82.1 A, beta = 104.4 degrees, while for the orthorhombic form a = 56.4 A, b = 74.6 A, c = 124.9 A. The asymmetric unit contains one dimer in both cases. The crystals diffract to 1.7 A resolution using synchrotron radiation. Preliminary data have been collected to 2.3 A on both crystal forms using a conventional X-ray source.     

Messages from ultrahigh resolution crystal structures

The ever growing availability of macromolecular crystal structures determined at atomic resolution has now reached a critical size, making it possible to obtain statistically unbiased data on both protein stereochemistry and the validity of the parameters used in their refinement. Besides the determination of the precise geometry of proteins and their active sites, high resolution structures have made it possible to check the application of normal mode calculations, to calculate charge density distributions and to analyze hydration shells around protein molecules. Even if only a few structures involve protein complexes, either with ligands or prosthetic groups, the information obtained in these cases is of great interest for obtaining the physical parameters of these interactions.     

X-Ray diffraction and high resolution NMR analysis of methyl D-glucopyranuronate derivatives

X-Ray diffraction and high resolution 1H and 13C NMR spectral data for methyl 2,3,4,-tri-O-acetyl-alpha-D-glucopyranuronate and methyl (allyl 2,3,4-tri-O-acetyl- beta-D-glucopyranosid)uronate are presented. Both compounds adopt the 4C1 conformation.     

New crystal forms and low resolution structure analysis of 20S proteasomes from bovine liver

20S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast. To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source. Here we report new crystal forms of 20S proteasomes from bovine liver and preliminary structure analysis of them. The crystals belong to the same space group but have different cell dimensions. One crystal (form I) belongs to space group P2(1)2(1)2(1) with unit cell dimensions of a = 124.8, b =197.4, c =323.8 A, and diffracts to 3.0 A resolution. The other crystal (form II) belongs to the same space group with a =115.1, b =205.6, c =316. 0 A, and diffracts to 4.0 A resolution. The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes.     

The atomic resolution crystal structure of atratoxin determined by single wavelength anomalous diffraction phasing

By using single wavelength anomalous diffraction phasing based on the anomalous signal from copper atoms, the crystal structure of atratoxin was determined at the resolution of 1.5 A and was refined to an ultrahigh resolution of 0.87 A. The ultrahigh resolution electron density maps allowed the modeling of 38 amino acid residues in alternate conformations and the location of 322 of 870 possible hydrogen atoms. To get accurate information at the atomic level, atratoxin-b (an analog of atratoxin with reduced toxicity) was also refined to an atomic resolution of 0.92 A. By the sequence and structural comparison of these two atratoxins, Arg(33) and Arg(36) were identified to be critical to their varied toxicity. The effect of copper ions on the distribution of hydrogen atoms in atratoxin was discussed, and the interactions between copper ions and protein residues were analyzed based on a statistical method, revealing a novel pentahedral copper-binding motif.     

用低温诱导法制备人高分辨染色体

目的：探讨非药物作用获得人高分辨染色体。方法：外周血淋巴细胞在３７℃中培养６６ｈ,入４℃冰箱４ｈ,再分别入３７℃恢复培养０、１０、２０、３０、４０、５０、６０ｍｉｎ,终止培养前以低浓度（０．０８μｇ／ｍｌ）秋水仙素处理培养物３０ｍｉｎ。结果：不恢复培养（０ｍｉｎ）或短时恢复培养（１０、２０ｍｉｎ）组分裂指数最高,晚前期、前中期和早中期染色体数目最多。结论：该法简便,不需药物诱导,便于推广。     

X-ray diffraction study of a new crystal form of the nucleosome core showing higher resolution

Crystals have been grown of intact (unproteolysed) nucleosome cores from a variety of sources. The unit cells are all very similar, with one core particle per asymmetric unit. The X-ray diffraction patterns extend to about 5 Å in the direction perpendicular to the plane of the flat particle, and to somewhat less than this in other directions. The arrangement of particles in the unit cell has been deduced from Patterson projection maps, which also indicate the presence of a particle dyad. The data are consistent with the earlier proposed model for the core particle in which the 146 base-pairs of DNA are wound in about $\text{1}\text{3}\text{4}$ turns of superhelix about a histone octamer core.High angle diffuse X-ray scattering from the crystals shows that the DNA of the core particle is in the B form. The anisotropy of the diffuse scattering shows that the DNA is not firmly fixed to the histone core all along the superhelix path, but only over limited regions whose location correlates well with those in which the DNA is differentially protected against nuclease digestion.     

The high resolution crystal structure of recombinant Crithidia fasciculata tryparedoxin-I.

Tryparedoxin-I is a recently discovered thiol-disulfide oxidoreductase involved in the regulation of oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata tryparedoxin-I in the oxidized state has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises residues 3 to 145 with 236 water molecules and has been refined using all data between a 19- and 1.4-A resolution to an R-factor and R-free of 19.1 and 22.3%, respectively. Despite sharing only about 20% sequence identity, tryparedoxin-I presents a five-stranded twisted beta-sheet and two elements of helical structure in the same type of fold as displayed by thioredoxin, the archetypal thiol-disulfide oxidoreductase. However, the relationship of secondary structure with the linear amino acid sequences is different for each protein, producing a distinctive topology. The beta-sheet core is extended in the trypanosomatid protein with an N-terminal beta-hairpin. There are also differences in the content and orientation of helical elements of secondary structure positioned at the surface of the proteins, which leads to different shapes and charge distributions between human thioredoxin and tryparedoxin-I. A right-handed redox-active disulfide is formed between Cys-40 and Cys-43 at the N-terminal region of a distorted alpha-helix (alpha1). Cys-40 is solvent-accessible, and Cys-43 is positioned in a hydrophilic cavity. Three C-H...O hydrogen bonds donated from two proline residues serve to stabilize the disulfide-carrying helix and support the correct alignment of active site residues. The accurate model for tryparedoxin-I allows for comparisons with the family of thiol-disulfide oxidoreductases and provides a template for the discovery or design of selective inhibitors of hydroperoxide metabolism in trypanosomes. Such inhibitors are sought as potential therapies against a range of human pathogens.     

Yeast tRNA(Asp)-aspartyl-tRNA synthetase complex: low resolution crystal structure

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125,000, and two molecules of its cognate tRNA (Mr = 24160) cocrystallize in the cubic space group I432 (a = 354 A). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D2O content in order to use the contrast variation method to distinguish between the protein and tRNA. The synthetase was first located at 40 A resolution using the 65% D2O neutron data (tRNA matched) tRNA molecules were found at 20 A resolution using both neutron and X-ray data. The resulting model was refined against 10 A resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.     

Three-dimensional electron diffraction of PhoE porin to 2.8 A resolution

A three-dimensional set of electron diffraction intensities of PhoE porin embedded in trehalose extending to 2.8 A resolution has been collected and analyzed. The strongest high-resolution intensities are distributed as a figure of revolution about the z*-axis and are located primarily in a resolution range of 4.5 A to 5.0 A. Within this region, centered near 4.8 A resolution the brightest intensities are clustered about inclination angles of 35 degrees and 0 degrees from the a*, b* plane. This distribution of intensities indicates that the beta-sheet in PhoE porin is arranged to form a cylinder-like structure that contains major populations of beta-sheet strands tilted an average of 35 degrees and 0 degrees with respect to the membrane plane normal. This cylindrical structure has been seen previously in the high-resolution projection map of PhoE as an elliptical ring of high density.     

Mechanism of the lamellar/inverse hexagonal phase transition examined by high resolution x-ray diffraction

For the first time the electron density of the lamellar liquid crystalline as well as of the inverted hexagonal phase could be retrieved at the transition temperature. A reliable decomposition of the d-spacings into hydrophobic and hydrophilic structure elements could be performed owing to the presence of a sufficient number of reflections. While the hydrocarbon chain length, d(C), in the lamellar phase with a value of 14.5 A lies within the extreme limits of the estimated chain length of the inverse hexagonal phase 10 A < d(C) < 16 A, the changes in the hydrophilic region vary strongly. During the lamellar-to-inverse hexagonal phase transition the area per lipid molecule reduces by approximately 25%, and the number of water molecules per lipid increases from 14 to 18. On the basis of the analysis of the structural components of each phase, the interface between the coexisting mesophases between 66 and 84 degrees C has been examined in detail, and a model for the formation of the first rods in the matrix of the lamellar phospholipid stack is discussed. Judging from the structural relations between the inverse hexagonal and the lamellar phase, we suggest a cooperative chain reaction of rod formation at the transition midpoint, which is mainly driven by minimizing the interstitial region.     

Crystals of cytochrome c-553 from Bacillus pasteurii show diffraction to 0.97 å resolution

We report here the purification and characterization of a c-type cytochrome present in the soluble fraction of the gram-positive, alkaliphilic, and highly ureolytic soil bacterium Bacillus pasteurii. The cytochrome is acidic (pI = 3.3), has a molecular mass of 9.5 kDa, and appears to dimerize in 150 mM ionic strength solution. The electronic spectrum is typical of a low-spin hexa-coordinated heme iron. Crystals of the protein in the oxidized state were grown by vapor diffusion at pH 5, by using 3.2 M ammonium sulfate as precipitant. Diffraction data at ultrahigh resolution (0.97 Å) and completeness (99.9%) have been collected under cryogenic conditions, by using synchrotron radiation. The crystals belong to the orthorhombic space group P2₁2₁2₁, with cell constants a = 37.14, b = 39.42, c = 44.02 Å, and one protein monomer per asymmetric unit. Attempts to solve the crystal structure by ab initio methods are in progress. Proteins 28:580–585, 1997 © 1997 Wiley-Liss, Inc.     

Low resolution models of self-assembled histone fibers from X-ray diffraction studies.

X-ray diffraction data from self-assembled histone fibers are presented for three systems: H4, H3-H4, and the four core histones H2A, H2B, H3 and H4. These data have been obtained under conditions of high ionic strength and high protein concentration which are thought to promote histone conformation similar to that found in intact chromatin. The low angle equatorial scattering (R less than .05 A-1) is analysed, and, with additional constraints imposed by electron microscopy data, four low resolution fibrillar models are derived. Two features common to all the possible models are a maximum outer diameter of approximately 60 A and a subfibril diameter of approximately 25 A. It is the interference of the protein subfibrils across a central region of low electron density - a 10 A "hole" - which gives rise to the characteristic diffraction peak at 36 A. Possible relationships of the models of the histone fibers to the structure of the histone component of chromatin are suggested.     

Tryptase from rat mast cells converts bovine prothrombin to thrombin

The effect of tryptase purified from rat peritoneal mast cells on bovine prothrombin was examined. Tryptase activated prothrombin, as evidenced by the increase in thrombin activity with a synthetic substrate, t-butyloxy-carbonyl-Val-Pro-Agr-4-methylcoumaryl-7-amide. The apparent Km value toward bovine prothrombin and the k_cat value were 2.3 μM and 46.3 s⁻¹, respectively. Studies on the time course of prothrombin activation by tryptase and by activated factor X (Xa), and analysis of the activation products on sodium dodecyl sulfate gel electrophoresis showed that the process of activation of prothrombin by tryptase was similar to that by Xa except that an intermediate of 67,000 daltons was formed.     

Plasmin converts factor X from coagulation zymogen to fibrinolysis cofactor

Known anticoagulant pathways have been shown to exclusively inhibit blood coagulation cofactors and enzymes. In the current work, we first investigated the possibility of a novel anticoagulant mechanism that functions at the level of zymogen inactivation. Utilizing both clotting and chromogenic assays, the fibrinolysis protease plasmin was found to irreversibly inhibit the pivotal function of factor X (FX) in coagulation. This was due to cleavage at several sites, the location of which were altered by association of FX with procoagulant phospholipid (proPL). The final products were approximately 28 and approximately 47 kDa for proPL-bound and unbound FX, respectively, which did not have analogues when activated FX (FXa) was cleaved instead. We next investigated whether the FX derivatives could interact with the plasmin precursor plasminogen, and we found that plasmin exposed a binding site only on proPL-bound FX. The highest apparent affinity was for the 28-kDa fragment, which was identified as the light subunit disulfide linked to a small fragment of the heavy subunit (Met-296 to approximately Lys-330). After cleavage by plasmin, proPL-bound FX furthermore was observed to accelerate plasmin generation by tissue plasminogen activator. Thus, a feedback mechanism localized by proPL is suggested in which plasmin simultaneously inhibits FX clotting function and converts proPL-bound FX into a fibrinolysis cofactor. These data also provide the first evidence for an anticoagulant mechanism aimed directly at the zymogen FX.     

Preparative scale, high resolution purification of low molecular weight DNA fragments

A commercially available continuous electroelution system has been used to separate and purify low molecular weight DNA fragments from polyacrylamide gels. This method has several advantages over previously reported methods for the recovery of DNA fragments from polyacrylamide gels. This technique, which gives very high recovery rates (80-95%), can be carried out on a relatively large scale and in a way that is not labour intensive. Data are presented for the purification of DNA fragments with molecular weights in the range 1-4 x 10(5) (200-700 base-pairs), although the method is also applicable to larger molecular weight DNA fragments, RNAs and proteins.