X-ray structure of the cyclomaltohexaicosaose triiodide inclusion complex provides a model for amylose-iodine at atomic resolution.

Cyclomaltohexaicosaose (CA26) is folded into two 1(2)/(3) turns long V-helices that are oriented antiparallel. Crystals of complexes of CA26 with NH(4)I(3) and Ba(I(3))(2) are brown and X-ray analyses show that I(3)(-) units are located in the approximately 5 A wide central channels of the V-helices. In the complex with NH(4)I(3), two CA26 molecules are stacked to form 2 x 1(2)/(3) turns long channels harbouring 3 I(3)(-) at 3.66-3.85 A inter I(3)(-) distance (shorter than van der Waals distance, 4.3 A), whereas in the Ba(I(3))(2) complex, CA26 are not stacked and only one I(3)(-) each fills the V-helices. Glucose...I contacts are formed with C5-H, C3-H, C6-H and (at the ends of the V-helices) with O6 in (+) gauche orientation. By contrast, O2, O3, O4 and O6 in the preferred (-) gauche orientation do not interact with I because these distances are >/=4.01 A and exceed the van der Waals I...O sum of radii by about 0.5 A except for one O2...I distance of 3.68 A near the end of one V-helix. Raman spectra indicate that the complexes share the presence of I(3)(-) with blue amylose-iodine.

Degradation pathway of the phosphonate ciliatine: crystal structure of 2-aminoethylphosphonate transaminase.

Phosphonates allow certain organisms to thrive in otherwise hostile environments, and 2-aminoethylphosphonate (AEP) is a precursor of many cellular phosphonates. AEP transaminase (AEPT) is an enzyme essential to phosphonate synthesis and degradation pathways. The crystal structure of AEP transaminase was determined by multiwavelength anomalous diffraction of 66 selenium atoms. The refined structure at 2.2 A resolution revealed an overall fold and active site location similar to those of the dimeric, two-domain structure of type I aminotransferases. The active site contains a cofactor, pyridoxal 5'-phosphate (PLP), and the product phosphonoacetaldehyde. Comparison with other type I aminotransferase structures shows that the PLP-protein interactions are conserved. Modeling of bound substrates and products reveals the structural basis for AEP recognition and the stereospecificity of proton elimination at the alpha-carbon and indicates conformational changes along the reaction pathway.

Effect of peracylation of beta-cyclodextrin on the molecular structure and on the formation of inclusion complexes: an X-ray study.

The molecular structures of peracylated beta-cyclodextrins (CDs)--heptakis(2,3,6-tri-O-acetyl)-beta-CD (TA), heptakis(2,3,6-tri-O-propanoyl)-beta-CD (TP), and heptakis(2,3,6-tri-O-butanoyl)-beta-CD (TB)--have been determined by single crystal X-ray structure analysis. Due to the lack of O2...O3' hydrogen bonds between adjacent glucose units of the peracylated CDs, the macrocycles are elliptically distorted into nonplanar boat-shaped structures. The glucose units are tilted with respect to the O4 plane to relieve steric hindrance between adjacent acyl chains. In TB, all glucose units adopt the common (4)C(1)-chair conformation and one butanoyl chain intramolecularly penetrates the cavity, whereas, in TA and TP, one glucose unit each occurs in (O)S(2)-skew-boat conformation and one acyl chain closes the O6 side like a lid. In each of the three homologous molecules the intramolecular self-inclusion and lidlike orientation of acyl chains forces the associated O5-C5-C6-O6 torsion angle into a trans-conformation never observed before for unsubstituted CD; the inclusion behavior of TA, TP, and TB in solution has been studied by circular dichroism spectroscopy with the drug molsidomine and several organic compounds. No inclusion complexes are formed, which is attributed to the intramolecular closure of the molecular cavity by one of the acyl chains.

X-ray structure of beta-cyclodextrin-2,7-dihydroxy-naphthalene.4.6 H(2)O: an unusually distorted macrocycle.

The inclusion complex beta-cyclodextrin.2,7-dihydroxynaphthalene.4.6 H(2)O crystallized in the monoclinic space group P2(1), with a=14.082(3), b=19.079(4), c=12.417(3) A, beta=109.28(3) degrees, V=3149.0(11) A(3), and Z=2. An X-ray study performed at room temperature shows that the crystal packing is of the herringbone type with one 2,7-dihydroxynaphthalene included completely in the beta-CD cavity, its long axis being oriented along the beta-CD molecular axis, and 4.6 water molecules are placed in the interstitial space. The beta-CD macrocycle is elliptically distorted, and the guest molecule is held in the hydrophobic beta-CD cavity by C-H...O and C-H...pi interactions.

Crystal structure of low-potential cytochrome c549 from Synechocystis sp. PCC 6803 at 1.21 A resolution.

The crystal structure of low-potential cytochrome c549, an extrinsic component of the photosystem II (PS II) from Synechocystis sp. PCC 6803, was obtained directly from single-wavelength 1.21 A resolution diffraction data. This is the first monodomain bis-histidinyl monoheme cytochrome c to be structurally characterized. The extended N-terminal region of c549 builds up a two-strand antiparallel beta-sheet in a hairpin motif, which extends through two molecules owing to crystal packing. Both peptide termini are involved in crystal contacts, which may explain their protrusion out of the globular fold. The C-terminus is preceded by a 9 A-long hydrophobic finger extending from a positively charged base and could be involved in PSII interactions, as well as a protruding negative patch built by a set of conserved acidic residues among c549 sequences.

Ab initio structure determination of the lantibiotic mersacidin.

The crystal structure of mersacidin, a potential novel antibiotic against methicillin- and vancomycin-resistant Staphylococcus aureus strains, has been determined by ab initio methods. Despite all crystals being merohedrally twinned, an accurate structural model with an R value of 13.4% has been obtained at atomic resolution. With six molecules in the asymmetric unit and no atom heavier than sulfur, the structure corresponds to a protein of 120 amino acids and is the largest approximately equal-atom unknown structure solved by direct methods. In the crystal, the molecule assumes a compact fold different from that found by NMR in solution. Comparison of the NCS-related molecules reveals regions of variable flexibility. The region highly homologous to the related antibiotic actagardine is very rigid and possibly defines an essential building block of this class of new antibacterial substances.

The crystal structure of N(10)-formyltetrahydrofolate synthetase from Moorella thermoacetica.

The structure was solved at 2.5 A resolution using multiwavelength anomalous dispersion (MAD) scattering by Se-Met residues. The subunit of N(10)-formyltetrahydrofolate synthetase is composed of three domains organized around three mixed beta-sheets. There are two cavities between adjacent domains. One of them was identified as the nucleotide binding site by homology modeling. The large domain contains a seven-stranded beta-sheet surrounded by helices on both sides. The second domain contains a five-stranded beta-sheet with two alpha-helices packed on one side while the other two are a wall of the active site cavity. The third domain contains a four-stranded beta-sheet forming a half-barrel. The concave side is covered by two helices while the convex side is another wall of the large cavity. Arg 97 is likely involved in formyl phosphate binding. The tetrameric molecule is relatively flat with the shape of the letter X, and the active sites are located at the end of the subunits far from the subunit interface.

High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence.

A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and structural consequences of truncating the binding loop residues to alanine. In addition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitution at position P(1) and an unrelated Met52Leu change. In spite of the binding loop modification, the affinity for trypsin is only 30 times lower than that of the wild-type protein. At pH 7.5 the protein can be crystallized on the time-scale of hours, yielding very stable crystals of a new (tetragonal) form of BPTI. Conventional source X-ray data collected to 1.4 A at room temperature allowed anisotropic structure refinement characterized by R=0.1048. The structure reveals all 58 residues, including the complete C terminus, which is in a salt-bridge contact with the N terminus. The Cys14-Cys38 disulfide bridge is observed in two distinct chiralities. This bridge, together with an internal water molecule, contributes to the stabilization of the binding loop. The Ala mutations have only an insignificant and localized effect on the binding loop, which retains its wild-type conformation (maximum deviation of loop C(alpha) atoms of 0.7 A at Ala13). Four (instead of the typical three) additional water molecules are buried in an internal cleft and connected to the surface via a sulfate anion. Three more SO(4)(2-) anions are seen in the electron density, one of them located on a 2-fold axis. It participates in the formation of a dimeric structure between symmetry-related BPTI molecules, in which electrostatic and hydrogen bonding interactions resulting from the mutated Lys15Arg substitution are of central importance. This dimeric interaction involves direct recognition loop-recognition loop contacts, part of which are hydrophobic interactions of the patches created by the alanine mutations. Another 2-fold symmetric interaction between the BPTI molecules involves the formation of an antiparallel intermolecular beta-sheet that, together with the adjacent intramolecular beta-hairpin loops, creates a four-stranded structure.

Crystal structure determination at 1.4 A resolution of ferredoxin from the green alga Chlorella fusca.

BACKGROUND: [2Fe-2S] ferredoxins, also called plant-type ferredoxins, are low-potential redox proteins that are widely distributed in biological systems. In photosynthesis, the plant-type ferredoxins function as the central molecule for distributing electrons from the photolysis of water to a number of ferredox-independent enzymes, as well as to cyclic photophosphorylation electron transfer. This paper reports only the second structure of a [2Fe-2S] ferredoxin from a eukaryotic organism in its native form. RESULTS: Ferredoxin from the green algae Chlorella fusca has been purified, characterised, crystallised and its structure determined to 1.4 A resolution - the highest resolution structure published to date for a plant-type ferredoxin. The structure has the general features of the plant-type ferredoxins already described, with conformational differences corresponding to regions of higher mobility. Immunological data indicate that a serine residue within the protein is partially phosphorylated. A slightly electropositive shift in the measured redox potential value, -325 mV, is observed in comparison with other ferredoxins. CONCLUSIONS: This high-resolution structure provides a detailed picture of the hydrogen-bonding pattern around the [2Fe-2S] cluster of a plant-type ferredoxin; for the first time, it was possible to obtain reliable error estimates for the geometrical parameters. The presence of phosphoserine in the protein indicates a possible mechanism for the regulation of the distribution of reducing power from the photosynthetic electron-transfer chain.

Advances in direct methods for protein crystallography.

Recent advances in ab initio direct methods have enabled the solution of crystal structures of small proteins from native X-ray data alone, that is, without the use of fragments of known structure or the need to prepare heavy-atom or selenomethionine derivatives, provided that the data are available to atomic resolution. These methods are also proving to be useful for locating the selenium atoms or other anomalous scatterers in the multiple wavelength anomalous diffraction phasing of larger proteins at lower resolution.

Ab initio solution and refinement of two high-potential iron protein structures at atomic resolution.

The crystal structure of the reduced high-potential iron protein (HiPIP) from Chromatium vinosum has been redetermined in a new orthorhombic crystal modification, and the structure of its H42Q mutant has been determined in orthorhombic (H42Q-1) and cubic (H42Q-2) modifications. The first two were solved by ab initio direct methods using data collected to atomic resolution (1.20 and 0. 93 A, respectively). The recombinant wild type (rc-WT) with two HiPIP molecules in the asymmetric unit has 1264 protein atoms and 335 solvent sites, and is the second largest structure reported so far that has been solved by pure direct methods. The solutions were obtained in a fully automated way and included more than 80% of the protein atoms. Restrained anisotropic refinement for rc-WT and H42Q-1 converged to R(1) = summation operator||F(o)| - |F(c)|| / summation operator|F(o)| of 12.0 and 13.6%, respectively [data with I > 2sigma(I)], and 12.8 and 15.5% (all data). H42Q-2 contains two molecules in the asymmetric unit and diffracted only to 2.6 A. In both molecules of rc-WT and in the single unique molecule of H42Q-1 the [Fe(4)S(4)](2+) cluster dimensions are very similar and show a characteristic tetragonal distortion with four short Fe-S bonds along four approximately parallel cube edges, and eight long Fe-S bonds. The unique protein molecules in H42Q-2 and rc-WT are also very similar in other respects, except for the hydrogen bonding around the mutated residue that is at the surface of the protein, supporting the hypothesis that the difference in redox potentials at lower pH values is caused primarily by differences in the charge distribution near the surface of the protein rather than by structural differences in the cluster region.

Ab initio structure solution of a dimeric cytochrome c3 from Desulfovibrio gigas containing disulfide bridges.

The 1.2 A resolution crystal structure of the 29 kDa di-tetrahaem cytochrome c3 from the sulfate reducing bacterium Desulfovibrio gigas was solved by ab initio methods, making this the largest molecule to be solved by this procedure. The actual refined model of the cysteine-linked dimeric molecule reveals that this molecule is very similar to the non-covalently linked symmetrical dimer of the di-tetrahaem cytochrome c3 from Desulfomicrobium norvegicum. Each monomer has the typical polypeptide fold, haem arrangement and iron coordination found for the tetrahaem cytochrome c3 molecules. The interface between the covalently linked monomers in the asymmetric unit of the crystal shows a pseudo two-fold arrangement, disturbed from symmetry by crystal packing forces. The fact that D. gigas contains a dimeric tetrahaem cytochrome with solvent accessible disulfide bridges and that this cytochrome specifically couples hydrogen oxidation to thiosulfate reduction in bacterial extracts provides an interesting aspect related to disulfide exchange reactions in this microorganism.

The 1.2 A crystal structure of hirustasin reveals the intrinsic flexibility of a family of highly disulphide-bridged inhibitors.

BACKGROUND: Leech-derived inhibitors have a prominent role in the development of new antithrombotic drugs, because some of them are able to block the blood coagulation cascade. Hirustasin, a serine protease inhibitor from the leech Hirudo medicinalis, binds specifically to tissue kallikrein and possesses structural similarity with antistasin, a potent factor Xa inhibitor from Haementeria officinalis. Although the 2.4 A structure of the hirustasin-kallikrein complex is known, classical methods such as molecular replacement were not successful in solving the structure of free hirustasin. RESULTS: Ab initio real/reciprocal space iteration has been used to solve the structure of free hirustasin using either 1.4 A room temperature data or 1.2 A low temperature diffraction data. The structure was also solved independently from a single pseudo-symmetric gold derivative using maximum likelihood methods. A comparison of the free and complexed structures reveals that binding to kallikrein causes a hinge-bending motion between the two hirustasin subdomains. This movement is accompanied by the isomerisation of a cis proline to the trans conformation and a movement of the P3, P4 and P5 residues so that they can interact with the cognate protease. CONCLUSIONS: The inhibitors from this protein family are fairly flexible despite being highly cross-linked by disulphide bridges. This intrinsic flexibility is necessary to adopt a conformation that is recognised by the protease and to achieve an optimal fit, such observations illustrate the pitfalls of designing inhibitors based on static lock-and-key models. This work illustrates the potential of new methods of structure solution that require less or even no prior phase information.

1.7 A structure of the stabilized REIv mutant T39K. Application of local NCS restraints.

The X-ray structure of the T39K mutant of the variable domain of a human immunoglobulin kappa light chain has been determined at room temperature to 1.7 A resolution with a conventional R factor of 0. 182. T39K crystallizes in the triclinic space group P1 [a = 35.4 (1), b = 40.1 (1), c = 43.1 (1) A, alpha = 66.9 (1), beta = 85.4 (1), gamma = 73.8 (1) degrees ]. The unit-cell contains two monomers, related by a non-crystallographic twofold axis. The use of a novel type of local non-crystallographic symmetry restraints on related isotropic displacement parameters and 1-4 distances as incorporated in the refinement program SHELXL improves the model and quality of the maps, but local differences between both monomers in areas subject to different packing contacts can still be observed. 12 overall anisotropic scaling parameters were refined. These may have compensated for the difficulties in accurately scaling single rotation axis image plate data from a triclinic crystal, because of the scarcity of common equivalent reflections. The final model has been used to perform a number of tests on anisotropic scaling, non-crystallographic symmetry, anisotropic refinement, determination of standard uncertainties and bulk solvent correction. It is remarkable that removal of the NCS restraints from the final model caused Rfree to increase. These tests clarify the strategies for optimum use of SHELXL for refinement at medium as opposed to atomic resolution.

Can anomalous signal of sulfur become a tool for solving protein crystal structures?

A general method for solving the phase problem from native crystals of macromolecules has long eluded structural biology. For well diffracting crystals this goal can now be achieved, as is shown here, thanks to modern data collection techniques and new statistical phasing algorithms. Using solely a native crystal of tetragonal hen egg-white lysozyme, a protein of 14 kDa molecular mass, it was possible to detect the positions of the ten sulfur and seven chlorine atoms from their anomalous signal, and proceed from there to obtain an electron-density map of very high quality.

V-Amylose at atomic resolution: X-ray structure of a cycloamylose with 26 glucose residues (cyclomaltohexaicosaose).

The amylose fraction of starch occurs in double-helical A- and B-amyloses and the single-helical V-amylose. The latter contains a channel-like central cavity that is able to include molecules, "iodine's blue" being the best-known representative. Molecular models of these amylose forms have been deduced by solid state 13C cross-polarization/magic angle spinning NMR and by x-ray fiber and electron diffraction combined with computer-aided modeling. They remain uncertain, however, as no structure at atomic resolution is available. We report here the crystal structure of a hydrated cycloamylose containing 26 glucose residues (cyclomaltohexaicosaose, CA26), which has been determined by real/reciprocal space recycling starting from randomly positioned atoms or from an oriented diglucose fragment. This structure provides conclusive evidence for the structure of V-amylose, as the macrocycle of CA26 is folded into two short left-handed V-amylose helices in antiparallel arrangement and related by twofold rotational pseudosymmetry. In the V-helices, all glucose residues are in syn orientation, forming systematic interglucose O(3)n...O(2)(n+l) and O(6)n...O(2)(n+6)/O(3)(n+6) hydrogen bonds; the central cavities of the V-helices are filled by disordered water molecules. The folding of the CA26 macrocycle is characterized by typical "band-flips" in which diametrically opposed glucose residues are in anti rather than in the common syn orientation, this conformation being stabilized by interglucose three-center hydrogen bonds with O(3)n as donor and O(5)(n+l), O(6)(n+l) as acceptors. The structure of CA26 permitted construction of an idealized V-amylose helix, and the band-flip motif explains why V-amylose crystallizes readily and may be packed tightly in seeds.

Structure and molecular modelling of protected dipeptide fragment (Boc-Phe-Leu-OBzl) of enkephalin.

The conformational characteristics of a flexible totally protected C-terminal dipeptide fragment (Boc-Phe-Leu-OBzl) of enkephalin are studied using X-ray data, molecular modelling and data retrieved from the Cambridge Structural Database. The dipeptide crystallizes with seven conformers in the asymmetric unit. C(27)H(36)N(2)O(5), T = 133 K, monoclinic, P2(1), a = 13.706 (3), b = 22.800 (3), c = 30.674 (5) Å, beta = 97.15 (3) degrees, V = 9511 (3) Å(3), Z = 14, D(c) = 1.145 Mg m(-3). Six of the seven molecules exhibit folded conformations with hydrophobic groups disposed at the opposite side of the peptide backbone. The characteristic Phi(1) and Psi(1) angles of the Phe residue and Phi(2) of the Leu fragment are in the allowed region defined in the Ramachandran diagram. However, they do not belong to the family of the lowest energy conformations. In the crystal, molecules are interconnected via N-H.O hydrogen bonds of peptide groups forming an infinite sheet similar to a parallel beta-sheet. Molecular dynamics simulations performed in vacuo reproduce the conformers and rotamers detected in the solid state.

Structure of balhimycin and its complex with solvent molecules.

Balhimycin is a naturally occurring glycopeptide antibiotic, related to vancomycin which acts by binding nascent bacterial cell-wall peptide ending in the sequence D-Ala-D-Ala. Crystals of balhimycin are monoclinic, space group P21, a = 20.48 (10), b = 43.93 (21), c = 27.76 (14) A, beta = 100.5 (5) degrees with four independent antibiotic molecules, three molecules of 2-methyl-2,4-pentanediol, two citrate ions, three acetate ions and 127.5 water molecules in the asymmetric unit. With an asymmetric unit larger than those of the smallest proteins and a solvent content of about 32%, the crystals have similar diffraction properties to those of small proteins. 27387 unique reflections were collected using synchrotron radiation. The structure was solved by a standard protein technique, the molecular-replacement method, using ureido-balhimycin as search model. The anisotropic refinement against all F2 data between 0.96 and 45 A converged to a conventional R value of 11.27% with R1= Sigma||Fo|-|Fc||/Sigma|Fo| for the 24623 data with I > 2sigma(I) and 12.58% for all 27387 data. The four monomers possess fairly similar conformations (r.m.s. deviation 0.7 A). Two antibiotic molecules form a tight dimer with antiparallel hydrogen bonds between the peptide backbone as well as between the vancosamine residues and the peptide backbone. In each of the two dimers, one binding pocket is occupied by a citrate ion and the other by an acetate ion. The dimer units are linked in the crystal by hydrogen bonds to form infinite chains.

X-ray structure of Tyr-D-Tic-Phe-Phe-NH2 (D-TIPP-NH2), a highly potent mu-receptor selective opioid agonist. Comparison with proposed model structures.

Tyr-D-Tic-Phe-Phe-NH2 (D-TIPP), a linear tetrapeptide containing the conformationally restricted Tic residue (tetrahydroisoquinoline-3-carboxylic acid), is an opioid agonist which exhibits high affinity and selectivity for the mu-receptor. Its conformational features have been studied using a combination, a solid-state (X-ray) and modeling (molecular mechanics and Monte Carlo simulations) methods. The results of the X-ray study showed two distinct conformers for D-TIPP, with the main differences lying in the orientation of the Tyr side-chain and the presence of both D-Tic(+) and D-Tic(-) conformations for the D-Tic residue. The peptide backbone is folded and stabilized by the formation of one intramolecular hydrogen bond. The modeling results also indicated a folded backbone for the peptide and both cis and trans conformers for the D-Tic residue are found in the lowest-energy structures. Comparison of the X-ray and modeling results shows many similarities especially around the D-Tic residue.

The bi-loop, a new general four-stranded DNA motif.

The crystal structure of the cyclic octanucleotide d contains two independent molecules that form a novel quadruplex by means of intermolecular Watson-Crick A.T pairs and base stacking. A virtually identical quadruplex composed of G.C pairs was found by earlier x-ray analysis of the linear heptamer d(GCATGCT), when the DNA was looped in the crystal. The close correspondence between these two structures of markedly dissimilar oligonucleotides suggests that they are both examples of a previously unrecognized motif. Their nucleotide sequences have little in common except for two separated 5'-purine-pyrimidine dinucleotides forming the quadruplex, and by implication these so-called "bi-loops" could occur widely in natural DNA. Such structures provide a mechanism for noncovalent linking of polynucleotides in vivo. Their capacity to associate by base stacking, demonstrated in the crystal structure of d(GCATGCT), creates a compact molecular framework made up of four DNA chains within which strand exchange could take place.