REFINEMENT OF THE STRUCTURE OF DESPENTAPEPTIDE (B26—30) INSULIN AT 1.5 RESOLUTION

The refinement of the structure of despentapeptide (B26-20) insulin (DPI) at 1.5 Aresolution is described in the present paper. Against the X-ray diffraction data of newlygrown crystals, the restrained parameter least-squares refinement of the structure alter-nated with model-rebuilding. The original 2.4A resolution model has been thoroughlyrefined and parts of it have been eonsiderably revised. The cadmium coordination struc-ture has been well determined, and appears to be a nearly ideal octahedron. After refine-ment 84 water oxygen atoms have been included in the final model; this gives us detailedinformation for studies on the water structure in the DPI crystal. The final crystal-lographic reliability index R value was 0.144 for 5678 unique observable reflexions (F_o great-er than 1.5σ (F_o)) in the 10-1.5A range; the refined model has a root-mean-squaredeviation from standard bond lengths of 0.04A.     

CRYSTAL STRUCTURE DETERMINATION OF DESHEPTAPEPTIDE (B24—B30)-INSULIN

Desheptapeptide (B24—B30)-insulin (DHPI), an essentially inactive insulin analog, is crystallized in space group P2_12_12_1 with two molecules in an asymmetric unit. The orientations of the molecules in the crystal cell have been determined by using Patterson search method at 6 resolution and the positions of the molecules are deduced from translation function calculation and R search at 3. resolution. After using the rigid body refinement (CORELS) further to refine the orientational and positional parameters as well as the initial energy restrained refinement (EREF) for the model, the crystallographic R valueis reduced to 0.384 at 3 resolution. The initial Fourier map shows that the B-chain N-terminal (B1—B8) and C-terminal (B20—B22)segments, compared with the native 2 zinc insulin, exhibit drastic conformational changes, but the three helices of B- and A-chains and their relative arrangement are essentially kept in DHPI.     

STRUCTURE OF (Trp)~(B1)-INSULIN AT 2RESOLUTION

The rhombohedral crystal structure of [Trp]~(B1)-insulin has been refined, using data to 2 and atomic coordinates of 2-zinc porcine insulin as starting model, and through the use of the restrained least-squares method, to an R value of 0.24. The result of refinement shows that in comparison with the 2-zinc insulin structure, some changes in local conformation occur, and are asymmetric for the two independent molecules related by a local two-fold axis. The obvious conformational changes are found to be in molecule 1, at its B-chain N-terminus and A13-residue region.     

STRUCTURE DETERMINATION OF THE SILVER CARP INSULIN CRYSTAL

After rotation search of an orthorhombic crystal form of Silver Carp insulin, based on the known porcine 2-Zn insulin structure, the Silver Carp insulin molecules were positioned in the unit cell by applying the translation function and R-factor search methods. The packing of the molecules in the cell is reasonably good. A preliminary refinement of this model at 3.5 resolution has been carried out through the use of restrained least-squares method. The segments of polypeptide chain, which were not involved in the refinement, could be visible in the electron density map calculated using the refined atomic parameters.     

STUDIES ON THE CRYSTAL STRUCTURE OF Al-(L-TRYPTOPHAN) INSULIN AT 2.1RESOLUTION

In order to study the biological effect of alterations to the N-terminus of the insulin A-chain, we have determined the crystal structure of Al-(L-Trp) insulin and discovered that it belongs to the trigohal system with space group R3. The parameters oof the unit cell are a=b=80.3A, c=37.5A. The model was adjusted and refined by using a stereochemically-restrained least squares program, assisted by manual revision of the model based on the difference Fourier map, to a final R-factor of 0.195. The main and side chains of both Al-(L-Trp) residues in the asymmetric unit are well ordered. It was found that the Al-Trp residue of molecule I occupied two distinct positions. We have proposed from the results of the three-dimensional structure that the 4-zinc insulin hexameric form is a stored state of insulin molecules in a conformation of low activity. The structural details of the insulin molecule and its structure and function relationship have also been discussed.     

THE CRYSTAL STRUCTURE OF SILVER CARP INSULIN AT MEDIUM RESOLUTION

It is an important way of surveying the structure-functionrelationship of insulin tostudy insulins from different species. Based on the structure model of an orthorhombic crys-tal obtained by the molecular replacement method, the crystallographic refinement of a hex-amer of silver carp insulin in an asymmetric unit has been carried out with 2.8A resolutiondata using the restrained least- squares method. The comparisons of insulin structures haveshown that the six silver carp insulin molecules have very similar but not identical three- di-mensional structures which are similar to the known 2Zn pig insulin structure but remarka-bly different in some local conformations.     

X-RAY STUDIES ON THE STRUCTURE OF DES-PENTAPEPTIDE INSULIN AT 2.4 RESOLUTION——THE DPI MOLECULE AND ITS STRUCTURAL RELATIONSHIP WITH INSULIN

The structure of the monomeric insulin analogue des(B26—B30) insulin is presented.; A detailed comparison with the 2Zn insulin structures shows that while there are some large changes in the structure, the basic secondary structural units maintain their integrity. The DPI structure is broadly similar to molecule Ⅰ in the 2Zn structure, and in this respect is like other crystal forms of insulin. In addition to changes on the surface of the structure there are some subtle but extensive changes in the heart of the molecule. The molecules are closely packed in the crystal with many and varied contacts, including a complex network of protein-cadmium interactions and a considerable number of water mediated contacts. The molecular surface has an unusually large number of hydrophobic groups which tend to cluster in a thick band running around the protein. The crystal structure is well ordered, indeed the clarity of some side chains and the definition of the water molecules is superior to that found in the mor     

REFINEMENT OF THE CRYSTAL STRUCTURE OF INSULIN AT 1.2 RESOLUTION

The crystal structure of 2 Zn porcine insulin has been refined at 1.2 resolution. The reciprocal space refinement was restrained by the incorporation of extensive chemical observations into the least squares equations. In addition to the non-hydrogen atoms, hydrogen atoms of protein, water molecules and the bulk solvent have been determined and refined. After two cycles of anisotropic refinement of non-hydrogen atoms of insulin, the final agreement factor was 0.128 for 20,005 reflexions (F_o>1σ(F_o)) in a spacing 1.2 and the root mean squares deviation from ideal covalent bond lengths was 0.021. On the electron density maps, the appearances of weight and shape of non-hydrogen atoms were very clear and reasonable. The anisotropic appearances of sulphur atoms could be seen clearly. After anisotropic refinement, the situation fit to the electron density was improved distinctly.     

The further crystallographic refinement of trichosanthin at 2.7A resolution and the comparison among the three molecular structures in two crystal forms

The three-dimensional structure of trichosanthin at 2.7A resolution has been improved further, by refitting one of the C-terminal tails, adjusting 16 residues in the molecular surface regions, discarding some water molecules with high B values, and adjusting weights during the further refinement. The R-factor has been reduced to 18.5% and the r.m.s deviations from ideal geometry are also improved. The structures of the two molecules in the monoclinic asymmetric unit and the only molecule in the orthorhombic asymmetric unit are compared with one another. The main-chain structures for most of the residues in the three molecules are substantially the same. However, the courses of the three C-terminal tails are completely different, and the intermolecular interactions resulting from the particular packing of the molecules in the crystals account for the differences. The strand Be-2 and the preceding B-turn in small domain show large r.m.s. deviations among the three molecules and they are also involved in i     

Studies on Long-acting Insulin:Crystal Structure of Arg-B31 Human Insulin at 2.0 Resolution

The crystal structure of Arg-B31 human insulin(ABHI), a long-acting insulin derivative, has been determined at 2.0 resolution by using X-ray diffraction analysis. The final crystallographic R factor of the structure model after the refinement is 0.189 with the bond length r. m. s deviation of 0.018 . The refined structure of ABHI showed that the conformation of B-chain C-terminal residues was more stable than that in the native molecule. A striking structural feature of ABHI was an additional ion pair formed between ArgB31 of molecule Ⅰ and Glu-B21 of molecule Ⅱ in a dimer, and three ionic bonds between the neighbouring molecules thereby appeared on the surface of ABHI hexamer.These secondary bonds generated by the insertion of the residue Arg-B31 should make the rate of dissociation of ABHI hexamer slow down when it was injected into the body and the property of protraction should be produced by a 'depot effect'. This ought to be the main structure basis of the prolonged action of ABHI. The results o     

X-RAY STUDIES ON THE STRUCTURE OF DES-PENTAPEPTIDE INSULIN AT 2.4 RESOLUTION——THE REFINEMENT OF THE STRUCTURE

The determination of an initial model for the crystal structure of des-pentapeptide in-sulin, using only the phase information obtained from the scattering of cadmium ions inthe native crystal, has been described. This paper describes the refinement of this modelagainst both the experimental X-ray observations and the a priori stereochemical observationsusing the method of restrained parameter least squares. The corrected and extended modelincluded not only all the non-hydrogen atoms of the protein and one cadmium ion, but alsosome 49 water molecules. Fourier syntheses using amplitudes of 2F_o-F_c, phase angles prob-abilistically combined from the anomalous scattering and model phase information, witheach term weighted according to its reliability gave clear results without detectable bias to-wards the current model. The final model has acceptable stereochemistry (root mean squaredeviation from ideal bond lengths 0.027 A), and predicts the observed X-ray scattering sat-isfactorily (R = 0.182 for all dat     

STUDIES ON THE CRYSTAL STRUCTURE OF Al-(D-TRYPTOPHAN) INSULIN

We have determined the crystal structure of Al-(D-Trp) insulin and discovered that it belongs to the trigonal system with space group R3. The parameters of the unit cell are a=b=78.6, c=50.0. A set of data for half a sphere reciprocal space to a spacing of 2.2 were collected. The model was adjusted and refined by using a step-by-step approach and a stereochemically-restrained least squares program, assisted by manual revision based on the difference Fourier maps, to a final R-factor of 0.218. The main and side chains of both Al-D-Trp residues in the asymmetric unit are well ordered. The packing of Al-(D-Trp) insulin in the unit cell, the conformational differences with other insulin structures and its structure and function relationship bave also been discussed.     

THE COURSE OF THE POLYPEPTIDE CHAIN OF TRICHOSANTHIN MOLECULE

The course of the polypeptide chain of Trichosanthin has been determined from the electron density map at 4 resolution. The structure belongs to α+β type. It contains eight α-helices (39% of total residues) and four β-sheets (32% of total residues) which are made up of thirteen β-strands. The α-helices are relatively in the centre of the molecule and surrounded by β-sheets. This is the characteristic feature of Trichosanthin structure. This kind of structural arrangement has not been reported before. The transformation matrix and translation vector, which superpose two molecules in one asymmetric unit, were obtained. The root mean square error is 1.31 for this superposition.     

Structural Insight into the Binding of Hypoxanthine with Purine Nucleoside Phosphorylase from Streptococcus mutants

Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway and an attractive target for drug design. The crystal structure of Streptococcus mutants purine nucleoside phosphorylase(Smu PNP) has been solved by molecular replacement at 1.80  resolution and refined to R factors of 19.9%/23.7%(Rcryst/Rfree) . Sequence alignment and structural comparison show that Smu PNP has more similarity with PNPs isolated from human and malarial sources than the bacterial PNPs. The structure complexed with hypoxanthine(HPA) and sulfate ion was solved at 2.24  resolution and refined to R factors of 21.6%/24.1%(Rcryst/Rfree) . It is interesting to note that the resulting electron density indicated the product,HPA,presents in the active site although inosine was included in the crystallization mixture with Smu PNP. Asn233 and Glu191 are the important residues for ligand binding and recognition. Comparison with PNPs from different species gives detailed information about binding of small molecules on the active site,which is important for the studies of enzymatic mechanism and rational design of specific inhibitors for PNPs.     

REFINED CRYSTAL STRUCTURE OF INSULIN AT 1.8A RESOLUTION——HYDROGEN BONDS AND BOUND WATER

The hydrogen bonds in insulin fall into three cases: the helical hydrogen bonds in α- or 3_(10)helices, the non-helical one formed by polar groups of insulin itself, and the hydrogen bondsformed between insulin and water. By using the information obtained, the results of a seriesof biochemical investigations on insulin analogs related to B-chain C-terminal peptide can beinterpreted and it can also be inferred that the complex behaviours of the aggregation ofinsulin may play a protective role for the unique conformation of the molecule. Water structure also appears in the refined model. About one third of the water in anasymmetric unit is hydrogen-bonded to insulin molecules or each other, which are referred toas bound water. The polar and charged groups of insulin all show the tendencies to bind towater molecules as many as possible, which is a significant factor for the stabilization of theunique conformation of the molecule. The binding way of water molecules to insulin mole-cules is also analysed.     

Crystal structure of the complex of trichosanthin with NADPH at 0.172 nm resolution

The orthorhombic crystal structure of the complex of trichosanthin with nicotinamide adenine dinucleotide phosphate has been determined by molecular replacement method using one of the molecules of the monoclinic crystal structure of trichosanthin at 0.27 nm resolution as the search model. The crystallographic refinement at 0.172 nm resolution led to a final R-factor of 17.4% with root-mean-square deviations of 0.0013 nm and 3.8 from the ideal bond lengths and bond angles, respectively. The quality of the structure, the polypeptide chain fold and the comparison of it with that of the monoclinic trichosanthin structure, the location of nicotinamide adenine dinucleotide phosphate, the active site structure as well as the solvent structure are described.     

Influence of temperature on the structure of liquid water

Molecular dynamics simulations have been carried out for liquid water at 7 different temperatures to understand the nature of hydrogen bonding at molecular level through the investigation of the effects of temperature on the geometry of water molecules. The changes in bond length and bond angle of water molecules from gaseous state to liquid state have been observed, and the change in the bond angle of water molecules in liquid against temperature has been revealed, which has not been seen in literature so far. The analysis of the radial distribution functions and the coordinate numbers shows that, on an average, each water molecule in liquid acts as both receptor and donor, and forms at least two hydrogen bonds with its neigbors. The analysis of the results also indicates that the water molecules form clusters in liquid.     

WATER STRUCTURE IN DESPENTAPEPTIDE (B26—B30) INSULIN CRYSTAL AT 1.5  RESOLUTION

The water structure in despentapeptide (B26-B30) insulin crystal is presented in this paper. Ac-cording to inspection of the final F_0-extended (2F_0-F_c) Fourier map, 81 water molecules (about twothirds of solvent) with electron density greater tLan 0.4 e/A~3 are included in the water model to bediscussed. In a hydrogen bond length range of 2.4--3.2 A, 51 water molecules, that is, 63% of thetotal 81, are hydrogen bonded to protein, of which 12 link the adjacent protein molecules through one-water bridges and more through two-water bridges. There is a tight water network in a long crevice be-tween protein molecules; two Cd atoms, like two piles, prop up the network through three water molecules(Cd ligands) at either end of the network, indicating that Cd ions and water networks play an importantrole in close packing of protein molecules in the crystal.     

Microanalytical Techniques Applied to Phase Identification and Measurement of Solute Redistribution at the Solid/Liquid Interface of Frozen Fe-4.3Ni Doublets

 A Fe-4.3Ni alloy has been solidified directionally by using the Bridgman system. The solidification conditions were chosen to obtain an oriented cellular structure of δ-ferrite. These are: a positive temperature gradient of about 60 K/cm and a growth rate of 6.6 μm/s. A change in these conditions can lead either to the formation of austenite or to the competitive growth of δ-ferrite/γ-austenite. The solid/liquid interface of δ-ferrite cells has been frozen and double instability has been revealed at the tip of the cells. The instability is described as the first harmonic wave of fundamental undulation, which appeared at the formerly planar solid/liquid interface. This means that a doublet structure is formed only with the imposed specific conditions of solidification. The Ni-solute redistribution after back-diffusion has been measured across the δ-ferrite doublet. Results of energy dispersive X-ray (EDX) measurements on the distribution of Ni and Fe correspond well to the theoretical prediction for redistribution developed especially for oriented structure formation (two dimensional solidification). Additionally, electron backscattered diffraction from the bulk Fe-4.3Ni alloy sample allowed us to determine the local structure, i.e. the distribution of single crystallite orientations in the microstructure. A unique correlation between fluctuations of the Ni-solute redistribution and crystalline orientations in the δ-ferrite doublets has been demonstrated. Moreover, a relationship between geometrical asymmetry of the doublets and solute redistribution has also been found.