Restrained Least-squares Refinement Research Articles

Anatomy of atrans–cispeptide transition during least-squares refinement of rubrerythrin

A detailed view is presented of the effects of one round of 20 cycles of restrained least-squares refinement of rubreythrin in which a trans peptide between Gly78 and Ile79 converts to a cis conformation automatically. While the omega angle for the peptide changes by nearly 180 degrees , the maximum shift in any atomic position is 1.32 Angstroms. The peptide converts by passing through a non-ideal structure containing a nearly linear C-N-C(alpha) bond angle. The overall motion is not possible for real or virtual molecular models with ideal bond lengths, angles and torsion angles. Strengthening the stereochemical bond length and bond angle restraints halts the structural change.

Determination by MAD-DM of the structure of the DNA duplex d[ACGTACG(5-BrU)]2 at 1.46 A and 100 K.

A four-wavelength MAD experiment on a new brominated octanucleotide is reported here. d[ACGTACG(5-BrU)], C77H81BrN30O32P7, Mr (DNA) = 2235, tetragonal, P43212 (No. 96), a = 43.597, c = 26.268 A, V = 49927.5 A3, Z = 8, T = 100 K, R = 10.91% for 4312 reflections between 15.0 and 1.46 A resolution. The self-complementary brominated octanucleotide d[ACGTACG(5-BrU)]2 has been crystallized and data measured to 1.45 A at both 293 K and a second crystal flash frozen at 100 K. The latter data collection was carried out to the same resolution at the four wavelengths 0.9344, 0.9216, 0.9208 and 0.9003 A, around the Br K edge at 0.92 A and the structure determined from a map derived from a MAD data analysis using pseudo-MIR methodology, as implemented in the program MLPHARE. This is one of the first successful MAD phasing experiments carried out at Sincrotrone Elettra in Trieste, Italy. The structure was refined using the data measured at 0.9003 A, anisotropic temperature factors and the restrained least-squares refinement implemented in the program SHELX96, and the helical parameters are compared with those previously determined for the isomorphous d(ACGTACGT)2 analogue. The asymmetric unit consists of a single strand of octamer with 96 water molecules. No countercations were located. The A-DNA helix geometry obtained has been analysed using the CURVES program.

Structure of human diferric lactoferrin refined at 2.2 A resolution.

The three-dimensional structure of the diferric form of human lactoferrin has been refined at 2.2 A resolution, using synchrotron data combined with a lower resolution (3.2 A) diffractometer data set. Following restrained least-squares refinement and model rebuilding the final model comprises 5330 protein atoms (691 residues), 2Fe(3+) and 2CO(3)(2-) ions, 469 solvent molecules and 98 carbohydrate atoms (eight sugar residues). Root-mean-square deviations from standard geometry are 0.015 A for bond lengths and 0.038 A for angle (1-3) distances, and the final crystallographic R-factor is 0.179 for all 39 113 reflections in the resolution range 8.0-2.2 A. A close structural similarity is seen between the two lobes of the molecule, with differences mainly in loops and turns. The two binding sites are extremely similar, the only apparent differences being a slightly more asymmetric bidentate binding of the carbonate ion to the metal, and a slightly longer Fe-O bond to one of the Tyr ligands, in the N-lobe site relative to the C-lobe site. Distinct differences are seen in the interactions made by two cationic groups, Arg210 and Lys546, behind the iron site, and these may influence the stability of the two metal sites. Analysis of interdomain and interlobe interactions shows that these are few in number which is consistent with the known flexibility of the molecule with respect to domain and lobe movements. Internal water molecules are found in discrete sites and in two large clusters (in the two interdomain clefts) and one tightly bound water molecule is present 3.8 A from the Fe atom in each lobe. The carbohydrate is weakly defined and has been modelled to a limited extent; two sugar residues of the N-lobe glycan and six of the C-lobe glycan. Only one direct protein-carbohydrate contact can be found.

Synthetic, structural and biological studies of the ubiquitin system: chemically synthesized and native ubiquitin fold into identical three-dimensional structures

The solid-phase chemical synthesis of ubiquitin produced a molecule with physicochemical properties similar to those of the natural protein. We have grown crystals of this synthetic ubiquitin and performed an X-ray analysis at 1.8 A resolution in order to compare the synthetic protein with the known natural structure. The crystals were isomorphous with those of the natural protein, the R-factor between them being 7.1%. Difference Fourier analysis shows that the synthetic and natural structures are indistinguishable. The co-ordinates of the natural ubiquitin (1UBQ) were used as the starting point for restrained least-squares refinement (TNT program) against the synthetic X-ray data. The refinement converged to R = 16.5% and the resulting model did not change when refined against natural ubiquitin X-ray data (R = 18.7%). From both the refinement and featureless difference Fourier synthesis, we conclude that the synthetic and natural protein structures are identical. A short discussion about the uses of proteins with 'non-standard' amino acid residues is included.

Accuracy and precision in protein crystal structure analysis: two independent refinements of the structure of poplar plastocyanin at 173 K.

The structure of the copper protein plastocyanin from poplar leaves (Populus nigra var. italica) at 173 K has been subjected to two independent refinements, using a single set of synchrotron X-ray data at 1.6 A resolution. Energy-restrained refinement using the program EREF resulted in lower root-mean-square deviations from ideal geometry (e.g. 0.011 A for bond lengths) but a higher residual R (0.153) than restrained least-squares refinement using the program PROLSQ (0.014 A, 0.132). Electron-density difference maps in both refinements provided evidence for disorder at some side chains and solvent atoms, and the PROLSQ refinement made allowance for this disorder. The number of solvent sites identified at the 4sigma(rho) level was 171 in the EREF refinement and 189 in the PROLSQ refinement; 159 of the solvent sites are common to both refinements within 1 A. The root-mean-square differences between the atomic positions produced by the two refinements are 0.08 A for C(alpha) atoms, 0.08 A for backbone atoms and 0.12 A for all non-H atoms (excluding six obvious outliers) of the protein molecule. The two sets of Cu-ligand bond lengths differ by up to 0.07 A, and the ligand-Cu-ligand angles by up to 7 degrees. At 173 K the volume of the unit cell is 4.2% smaller than at 295 K. Greater order in the solvent region is indicated by the location of 79 more solvent sites, the identification of extensive networks of hydrogen-bonded rings of solvent molecules, and a general decrease in the thermal parameters. Within the unit cell, the protein molecules are significantly translated and rotated from their positions at ambient temperature. An important structural change at low temperature is a 180 degrees flip of the peptide group at Ser48-Gly49. Nearly all other significant differences between the structures of the protein at 173 and 295 K occur at exposed side chains. If the backbone atoms in the 173 and 295 K structures are superposed, excluding atoms involved in the peptide flip, the root-mean- square difference between the positions of 393 atoms is 0.25 A. Two internal water molecules, not included in previous descriptions of poplar plastocyanin, have been located. The plastocyanin Cu-site geometry at 173 K is not significantly different from that at 295 K. If plastocyanin undergoes a change in Cu-site geometry at low temperature, as has been suggested on the basis of resonance Raman spectroscopic evidence, then the change is not detected within the limits of precision of the present results.

Refined Structure of Trichosanthin at 1. 73 A Resolution

An X-ray reflection data set of the orthorhombic crystal of Trichosanthin(TCS) at 1. 73 A resolution had been collected using the area detector. We determined TCS crystal structure by the molecular replacement method using the data of the known α-momorcharin model and TCS intensities , and then TGS structure refinement at 1. 73 A resolution was performed with the restrained least-squares refinement. A final R-factor of 0.186 was obtained with a model obeying standard geometry within 0. 013A in bond lengths and 2. 48° in bond angles. The final model contains 133 solvent molecules in the asymmetric unit. This paper gives a detailed description of TCS molecular structure, temperature factors, hydrogen bonds, bound water, the distribution of conserved residues and the interactions between the TCS molecules. The hydrogen bond between the hydroxyl group of conserved residue 14Tyr and O157 plays an important role in maintaining the active site conformation. Conserved residue 160Glu and its conformation at the active site play a key role in the catalytic activity.

The faulted structures of [WCl2(PhC2Ph)(CH2=CH2)(PMe3)2] and [WCl2(PhC2Ph)2(PMe3)2]

The crystal structures of dichloro(diphenyl-η 2 -acetylene)(η 2 -ethylene)bis(trimethylphosphine)tungsten(IV) and dichlorobis(diphenyl-η 2 -acetylene)bis(trimethylphosphine)tungsten(IV) are described. The crystal structures are unusual in that they contain stacking faults leading to disordered structures. This has necessitated constrained restrained leastsquares refinement

Crystal structure determination and refinement at 2.3-A resolution of the lentil lectin.

We report on the X-ray structure determination of the orthorhombic crystal form of lentil lectin by molecular replacement using the pea lectin coordinates as a starting model. The structure was refined at 2.3-A resolution with a combination of molecular dynamics refinement and classical restrained least-squares refinement. The final R value for all data Fo > 1 sigma (Fo) between 7.0- and 2.3-A resolution is 0.164%, and deviations from ideal bond distances are 0.014 A. The C-terminus of the beta-chain proved to be 23 amino acids longer than found in previous studies. This together with several inconsistencies between the previously determined amino acid sequence and the observed electron density forced a redetermination of the amino acid sequence of the protein. The overall structure is very similar to that of pea lectin and isolectin I of Lathyrus ochrus, the most prominent deviations being confined to loop regions and the regions of intermolecular contact. The largest difference between the pea and lentil lectin monomers is situated in the loop region of amino acids 73-79 of the beta chain. There are no significant differences between the two crystallographic independent lentil lectin monomers in the asymmetric unit. The model includes 104 well-defined water molecules, of which a significant number have a counterpart in the pea lectin structure. As for the other legume lectins, each lentil lectin monomer contains one calcium ion in a highly conserved environment. On the contrary, the manganese binding sites are distorted with respect to the pea lectin and concanavalin A structures. The Asp beta 121 side chain apparently does not ligate the Mn2+ ion. This difference is consistent in both lentil lectin monomers and agrees with earlier solution studies. Possible implications for oligosaccharide binding are discussed.

Molecular dynamics in refinement against fiber diffraction data

The molecular dynamics (MD) method has been adapted for refinement of the structures of helical macromolecular aggregates aginst X-ray fiber diffraction data. To test the effectiveness of the method, refinements of the tobacco mosaic virus structure were carried out against a set of simulated fiber diffraction intensities using the MD method as well as the conventional restrained least-squares (RLS) method. The MD refinement converged to a very low R factor and produced a structure with generally satisfactory sterochemistry, while the RLS refinemnt was trapped at a local energy minimum with a larger R factor. Results suggest that the effective experimental radius of convergence of the MD method is significantly greater than that of the RLS method. Even when the initial structure is too far from the true structure to allow direct refinement, the MD method is able to find local minima that resemble the true structure sufficiently to allow improved phasing and thus lead to interpretable difference maps for model rebuilding.

Refinement of Recombinant Oncomodulin at 1·30 Å Resolution

A refinement of the oncomodulin crystal structure at 1·30 Å resolution has been carried out with X-ray data from the recombinant protein. The crystallographic R-factor values are 0·169 for 19,995 reflections in the range 6·0 to 1·30 Å, which were used for the restrained least-squares refinement, and 0·176 for 20,186 observed reflections in the range 10·0 to 1·30 Å. This high resolution refinement has enabled us to make more definitive statements about the molecular structure than was possible heretofore. The present model includes residues 1 to 108, the two Ca2+ of the CD and EF loops, two intermolecular Ca2+, and 103 water molecules per oncomodulin molecule. The electron density maps indicate disordered orientations for ten residues on the hydrophilic surface of the molecule. The pattern of molecular aggregation via intermolecular Ca2+, which occurs in the native rat oncomodulin structure, is also present in the recombinant oncomodulin structure. The Cys18 side-chain is not in a position that would be easily accessible for molecular dimerization via a disulphide bond. The substitution of Glu59, which is preserved in all the determined species of parvalbumin, by Asp59 in oncomodulin seems to break a stabilizing hydrogen bond in the CD loop and render the main-chain in positions 59 to 60 somewhat unstable. This instability in the CD loop, and the strong tendency of oncomodulin for molecular aggregation via intermolecular Ca2+, appear to be the two outstanding features that may account for oncomodulin's biological peculiarities.

Structure of the bovine eye lens protein gammaB(gammaII)-crystallin at 1.47 A.

The molecular structure of calf gammaB-crystallin (previously called gammaII), a lens-specific protein, has been refined to a crystallographic R factor of 18.1% for all reflection data, between 8.0 and 1.47 A, 25 959 hkl measured at 293 (1) K. 230 water molecules have been defined by difference Fourier techniques and included in a restrained least-squares refinement. Difference Fourier maps clearly indicated the presence of multiple sites for the sulfur atoms of Cys 18 and Cys 22 which were therefore given coupled second-site occupancies during the refinement. The sulfur atom in the major position of Cys 22 is in the reduced state. Either of the Cys 18 sites can form a high-energy disulfide bridge with the minor position of Cys 22. The position of the carboxy terminus and many other surface side chains have been further defined including the RGD signal peptide. The hydration of the backbone and the interdomain region has been analysed. 27 water molecules make extensive contacts to a single protein molecule and thus contribute to its stability.

Z-DNA structure of a modified DNA hexamer at 1,4-.ANG. resolution: aminohexyl-5'-d(pCpGp[Br5C]pGpCpG)

Oligonucleotides with modification at the 5'-end have been used for various biochemical applications. As a first step to better assess the effects of those modifications on DNA conformation, we determined at 1.4-A resolution the left-handed Z-DNA structure of a DNA hexamer, aminohexyl-5'-d(pCpGp[br5C]pGpCpG), by X-ray diffraction analysis. This hexamer was crystallized in the monoclinic C2 (a = 51.13 A, b = 18.44 A, c = 34.67 A, and beta = 120.9 degrees) space group. Its structure has been refined by the restrained least-squares refinement to a final R factor of 0.164 using 3727 [> 2.0 sigma (F)] observed reflections. The overall conformation of the double helix resembles that of the canonical Z-DNA. The terminal 5'-phosphate groups of the dC residues adopt conformations (beta approximately 180 degrees and gamma approximately 60 degrees) similar to phosphodiester's conformation of the internal dC residues. Two types of interhelical stackings are observed, one of which may serve as a model for a single-strand nick in the backbone of DNA double helix. A barium ion is found to bridge two side-by-side Z-DNA helices by coordinating to the O6 and N7 atoms of two guanines simultaneously. This "cross-linking" ability of barium ion may be a useful property in promoting the reversible aggregation of nucleic acids.

タンパク質結晶構造解析計算のベンチマークテスト

The CPU benchmark program for crystallographic computing is proposed by Dr.P.E.Bourne. The program consists of a restrained least-squares refinement using the programs PROTIN and PROLSQ for a small (545 atoms, 3590 diffraction maxima) and a large (6864 atoms, 19172 diffraction maxima) problem. The program was tested on more than 50 workstations and summarized. The result is helpful for (protein) crystallographers who will have a plan to buy a workstation. The program is available from the author by postal mail or anonymous ftp.

Proline cis-trans isomers in calbindin D 9k observed by X-ray crystallography

In a structure of recombinant bovine calbindin D 9k, determined crystallographically to 1.6 Å resolution, a proline in mixed, approximately equally populated, cis and trans conformation is observed. Isomers of this kind have not been reported in structure determinations of calbindin D 9k to 2.3 Å resolution or in any other crystallographically determined protein structure. The cis-trans isomerization occurs at the peptide bond between Gly42 and Pro43, which is in agreement with results from two-dimensional 1H nuclear magnetic resonance spectroscopy experiments on solutions of calbindin D 9k. Alternative backbone stretches have been modeled and refined by stereochemical restrained least-squares refinement for the segment Lys41 to Pro43. The final R-value was 0.188. The structural perturbations accompanying the cis-trans isomerization are found to be very localized. The largest positional differences are observed at residue Gly42, in which the alternative positions of the oxygen atom are 3.6 Å apart.

Structure of porcine insulin cocrystallized with clupeine Z

The crystal structure of NPH-insulin, pig insulin cocrystallized with zinc, m-cresol and protamine, has been solved by molecular replacement and refined using restrained least-squares refinement methods. The final crystallographic R factor for all reflections between 2 and 10 A is 19.4%. The insulin molecules are arranged as hexamers with two tetrahedrally coordinated Zn atoms in the central channel and one m-cresol bound to each monomer near His B5. One protamine binding site has been unequivocally identified near a dimer-dimer interface, although most of the polypeptide is crystallographically disordered. The conformation of the insulin moiety and the structural differences between the three unique monomers have been analysed. The zinc and m-cresol environments are described and the nature of the protamine binding site is outlined.

Structure and molecular model refinement of Aspergillus oryzae (TAKA) α-amylase: an application of the simulated-annealing method

Monoclinic crystals of a neutral alpha-amylase from Aspergillus oryzae, containing three molecules in the asymmetric unit, have been reported previously and studied at 3 A resolution [Matsuura, Kunusoki, Harada & Kakudo (1984). J. Biochem. 95, 697-702]. Here we report the solution of the structure of this enzyme in a different crystal form (space group P2(1)2(1)2(1), a = 50.9, b = 67.2, c = 132.7 A), with only one molecule in the asymmetric unit. The structure was solved by the molecular replacement method, using a model of acid alpha-amylase from a related fungus A. niger [Brady, Brzozowski, Derewenda, Dodson & Dodson (1991). Acta Cryst. B47, 527-535]. Conventional least-squares crystallographic refinement failed to converge in a satisfactory manner, and the technique of molecular dynamics in the form of the XPLOR package [Brunger (1988). XPLOR Manual. Yale Univ., USA] was used to overcome the problem. A large rigid-body type movement of the C-terminal domain was identified and accounted for. The final round of restrained least-squares refinement (at 2.1 A resolution) including 3675 protein atoms and 247 water molecules resulted in a conventional crystallographic R factor of 0.183 and an atomic model which conforms well to standard stereochemical parameters (standard deviation of bond lengths from their expected values is 0.028 A, while that for planar groups is 0.029 A).

X-ray analyses of aspartic proteinases: III. Three-dimensional structure of endothiapepsin complexed with a transition-state isostere inhibitor of renin at 1·6 Å resolution

The aspartic proteinase, endothiapepsin (EC 3.4.23.6), was complexed with a highly potent renin inhibitor, H-261 (t-Boc-His-Pro-Phe-His-LeuOHVal-Ile-His), where OH denotes a hydroxyethylene (-(S) CHOH-CH2-) transition-state isostere in the scissile bond surrogate. Crystals were grown in a form that has the same space group P2(1) as the uncomplexed enzyme, but with a 10 A decrease in the length of the alpha-axis and a 13 degrees decrease in the beta-angle. X-ray data have been collected to a resolution of 1.6 A. The rotation and translation parameters defining the position of the enzyme in the unit cell were determined previously using another enzyme-inhibitor complex that crystallized isomorphously with that of H-261. The molecule was refined using restrained least-squares refinement and the positions of non-hydrogen atoms of the inhibitor and water molecules were defined by difference Fourier techniques. The enzyme-inhibitor complex and 322 water molecules were further refined to a crystallographic R-factor of 0.14. Apart from a small rigid group rotation of a domain comprising residues 190 to 302 and small movements in the flap, there is little difference in conformation between the complexed and uncomplexed forms of the enzyme. The inhibitor is bound in an extended conformation along the active site cleft, and the hydroxyl group of the hydroxyethylene moiety is hydrogen-bonded to both catalytic aspartate carboxylates. The complex is stabilized by hydrogen bonds between the main-chain of the inhibitor and the enzyme. All side-chains of the inhibitor are in van der Waals' contact with groups in the enzyme and define a series of specificity pockets along the active site cleft. The study provides useful clues as to how this potent renin inhibitor (IC50 value of 0.7 x 10(-9) M) may bind renin. In particular it defines the interactions of the hydroxyethylene transition-state isostere with the enzyme more precisely than has been previously possible and therefore provides a useful insight into interactions in the transition state complex.

Refined structure of dienelactone hydrolase at 1.8A˚

The structure of dienelactone hydrolase (DLH) from Pseudomonus sp. B13, after stereochemically restrained least-squares refinement at 1.8A˚resolution, is described. The final molecular model of DLH has a conventional R value of 0.150 and includes all but the car☐yl-terminal three residues that are crystallographically disordered. The positions of 279 water molecules are included in the final model. The root-mean-square deviation from ideal bond distances for the model is 0.014A˚and the error in atomic co-ordinates is estimated to be 0.15A˚. DLH is a monomeric enzyme containing 236 amino acid residues and is a member of the β-ketoadipate pathway found in bacteria and fungi. DLH is an α/β protein containing seven helices and eight strands of β-pleated sheet. A single 4-turn 3 10-helix is seen. The active-site Cys123 resides at the N-terminal end of an α-helix that is peculiar in its consisting entirely of hydrophobic residues (except for a C-terminal lysine). The β-sheet is composed of parallel strands except for strand 2, which gives rise to a short antiparallel region at the N-terminal end of the central β-sheet. The active-site cysteine residue is part of a triad of residues consisting of Cys123, His202 and Asp171, and is reminiscent of the serine/cysteine proteases. As in papain and actinidin, the active thiol is partially oxidized during X-ray data collection. The positions of both the reduced and the oxidized sulphur are described. The active site geometry suggests that a change in the conformation of the native thiol occurs upon diffusion of substrate into the active site cleft of DLH. This enables nucleophilic attack by the γ-sulphur to occur on the cyclic ester substrate through a ring-opening reaction.

The use of single-wavelength anomalous scattering to solve the crystal structure of a gramicidin A/caesium chloride complex

Single-wavelength Cu K alpha anomalous scattering has been used to determine the structure of a crystalline complex of gramicidin A and caesium chloride. The asymmetric unit in these crystals, with space group P2(1)2(1)2(1) and a = 32.118 (6), b = 52.103 (12), c = 31.174 (7) A, contains four independent monomers (two dimers) of the pentadecapeptide. This structure falls in an intermediate size range for which direct methods and multiple isomorphous replacement are generally not successful for obtaining phase information. However, using the Bijvoet differences and the partial structure of the caesium atoms which have been incorporated in the crystals, it has been possible to obtain information on this crystal form. Because the caesium atoms dominate the scattering of these crystals, inclusion of the Friedel mate information in the restrained least-squares refinement has been essential. These studies extend the utility of single-wavelength anomalous-scattering phase determination to a macromolecular structure in which the partial structure of the anomalous scatterer is large.

Crystallographic refinement and structure of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum at 1.7 Å resolution

The amino acid sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum has been fitted to the electron density maps. The resulting protein model has been refined to a nominal resolution of 1.7 Å using the constrained-restrained least-squares refinement program of Sussman and the restrained least-squares refinement program of Hendrickson & Konnert. The crystallographic refinement, based on 76,452 reflections with F >σ(F) in the resolution range 5.5 to 1.7 Å resulted in a crystallographic R-factor of 18.0%. The asymmetric unit contains one dimeric ribulose-1,5-biphosphate carboxylase molecule, consisting of 869 amino acid residues and 736 water molecules. The geometry of the refined model is close to ideal, with root-mean-square deviations of 0.018 Å in bond lengths and 2.7 ° in bond angles. Two loop regions, comprising residues 54 to 63 and 324 to 335, and the last ten amino acid residues at the C terminus are disordered in our crystals. The expected trimodal distribution is obtained for the side-chain χ1-angles with a marked preference for staggered conformation. The hydrogen-bonding pattern in the N-terminal β-sheet and the parallel sheet in the βα-barrel is described. A number of hydrogen bonds and salt bridges are involved in domain-domain and subunit-subunit interactions. The subunit-subunit interface in the dimer covers an area of 2800 Å2. Considerable deviations from the local 2-fold symmetry are found at both the N terminus (residues 2 to 5) and the C terminus (residues 422 to 457). Furthermore, loop 8 in the ββ-barrel domain has a different conformation in the two subunits. A number of amino acid side-chains have different conformations in the two subunits. Most of these residues are located at the surface of the protein. An analysis of the individual temperature factors indicates a high mobility of the C-terminal region and for some of the loops at the active site. The positions and B-factors for 736 solvent sites have been refined (average B: 45·9 Å2). Most of the solvent molecules are bound as clusters to the protein. The active site of the enzyme, especially the environment of the activator Lys191 in the non-activated enzyme is described. Crystallographic refinement at 1.7 Å resolution clearly revealed the presence of a cis-proline at the active site. This residue is part of the highly conserved region Lys166-Pro167-Lys168.