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The 109 kDa quinohemoprotein amine dehydrogenase (QHNDH) from Paracoccus denitrificans contains a novel redox cofactor, cysteine tryptophylquinone (CTQ). This cofactor is derived from a pair of gene-encoded amino acids by post-translational modification and was previously identified and characterized within an 82-residue subunit by chemical methods and crystallographic analysis at 2.05 Å resolution. It contains an orthoquinone moiety bound to the indole ring and catalyzes the oxidation of aliphatic and aromatic amines through formation of a Schiff-base intermediate involving one of the quinone O atoms. This paper reports the structural analysis of the complex of QHNDH with the enzyme inhibitor phenylhydrazine determined at 1.70 Å resolution. The phenylhydrazone product is attached to the C6 position, identifying the O6 atom of CTO as the site of Schiff base formation as postulated by analogy to another amine-oxidizing enzyme, methylamine dehydrogenase. Furthermore, the inner N atom closest to the phenyl ring of phenylhydrazine forms a hydrogen bond to γASP33 in the complex, lending support to the hypothesis that this residue serves as the active-site base for proton abstraction during catalysis. [ABSTRACT FROM AUTHOR]

Crystallization is a unique process that occurs at the expense of entropy, including the conformational entropy of surface residues, which become ordered in crystal lattices during formation of crystal contacts. it could therefore be argued that epitopes free of amino acids with high conformational entropy are more thermodynamically favorable for crystal formation. For a protein recalcitrant to crystallization, mutation of such surface amino acids to residues with no conformational entropy might lead to enhancement of crystallization. This paper reports the results of experiments with an important cytosolic regulator of GTPases, human RhoGD1, in which lysine residues were systematically mutated to alanines. Single and multiple mutations were introduced into two different variants of RhoGD1, Nδ23 and Nδ66, in which the first 23 and 66 residues, respectively, were removed by recombinant methods. in total, 13 single and multiple mutants were prepared and assessed for crystallization and all were shown to crystallize using the Hampton Research Crystal Screens I and II, in contrast to wild-type Nδ23 and Nδ66 RhoGD1 which did not crystallize. Four crystal structures were solved (the triple mutants Nδ23:K135,138,141A and Nδ66:K135,138,141A, and two single mutants Nδ66:K113A and Nδ66:K141A) and in three cases the crystal contacts of the new lattices were found precisely at the sites of mutations. These results support the notion that it is, in principle, possible to rationally design mutations which systematically enhance proteins' ability to crystallize. [ABSTRACT FROM AUTHOR]

Human stuttering is a simple example of the repetition of sounds or symbols, sometimes associated with single letters, and may be used to illustrate the amazing repetition of amino acids (symbolized by a letter, e.g. W) in proteins. A survey of available databases with highly improbable strings of single amino acids is tabulated. This paper concludes with a challenge to the crystallographic community to probe the structural origins of the structure-function relationship in this neglected area. When nature stutters, we should pay attention. [ABSTRACT FROM AUTHOR]

Chemotaxis, mediated by methyl-accepting chemotaxis protein (MCP) receptors, plays an important role in the ecology of bacterial populations. This paper presents the first crystallographic analysis of the structure and ligand-induced conformational changes of the periplasmic tandem Per-Arnt-Sim (PAS) sensing domain (PTPSD) of a characterized MCP chemoreceptor. Analysis of the complex of the Campylobacter jejuni Tlp3 PTPSD with isoleucine (a chemoattractant) revealed that the PTPSD is a dimer in the crystal. The two ligand-binding sites are located in the membrane-distal PAS domains on the faces opposite to the dimer interface. Mutagenesis experiments show that the five strongly conserved residues that stabilize the main-chain moiety of isoleucine are essential for binding, suggesting that the mechanism by which this family of chemoreceptors recognizes amino acids is highly conserved. Although the fold and mode of ligand binding of the PTPSD are different from the aspartic acid receptor Tar, the structural analysis suggests that the PTPSDs of amino-acid chemoreceptors are also likely to signal by a piston displacement mechanism. The PTPSD fluctuates between piston (C-terminal helix) `up' and piston `down' states. Binding of an attractant to the distal PAS domain locks it in the closed form, weakening its association with the proximal domain and resulting in the transition of the latter into an open form, concomitant with a downward (towards the membrane) 4 Å piston displacement of the C-terminal helix. In vivo, this movement would generate a transmembrane signal by driving a downward displacement of the transmembrane helix 2 towards the cytoplasm. [ABSTRACT FROM AUTHOR]

The dra gene cluster of uropathogenic strains of Escherichia coli produces proteins involved in bacterial attachment to and invasion of the eukaryotic host tissues. The crystal structure of a construct of E. coli DraD possessing an additional C-terminal extension of 13 amino acids, including a His6 tag, has been solved at a resolution of 1.05 Å. The protein forms symmetric dimers through the exchange of the C-terminal ,β-strands, which participate in the immunoglobulin-like β-sandwich fold of each subunit. This structure confirms that DraD is able to act as an acceptor in the donor-strand complementation mechanism of fiber formation but, in contrast to DraE adhesin, its native sequence does not have a donor strand; therefore, DraD can only be located at the tip of the fiber. [ABSTRACT FROM AUTHOR]

Deletion of the N-terminal 31 amino acids from the coat protein (CP) of Sesbania mosaic virus (SeMV) results in the formation of T = 1 capsids. The X-ray crystal structure of CP-NΔ31 mutant capsids reveals that the CP adopts a conformation similar to those of other T = 1 mutants. The 40 N-terminal residues are disordered in CP-NA31. The intersubunit hydrogen bonds closely resemble those of the native capsid. The role of water molecules in the SeMV structure has been analyzed for the first time using the present structure. As many as 139 of the 173 waters per subunit make direct contacts with the protein atoms. The water molecules form a robust scaffold around the capsid, stabilize the loops and provide integrity to the subunit. These waters constitute a network connecting diametrically opposite ends of the subunit. Such waters might act as nodes for conveying signals for assembly or disassembly across a large conformational space. Many water-mediated interactions are observed at various interlaces. The twofold interlace, which has the smallest number of protein-protein contacts, is primarily held by water-mediated interactions. The present structure illuminates the role of water molecules in the structure and stability of the capsid and points out their possible significance in assembly. [ABSTRACT FROM AUTHOR]

The Ketch repeat is a common sequence motif in eukaryotic genomes and is approximately 50 amino acids in length. The structure of the Ketch domain of the human Kelch protein has previously been determined at 1.85 Å, showing that each Ketch repeat forms one btade of a six-bladed β-propeller. Here, use of 1.35 Å SAD data for de novo structure determination of the Ketch domain and for refinement at atomic resolution is described. The high quality and resolution of the diffraction data and phase information allows a detailed analysis of the rote of solvent in the structure of the Ketch repeat. Ten structurally conserved water molecules are identified in each blade of the Ketch β-propeller. These appear to play distinct structural roles that include lining the central channel of the propeller, interacting with residues in toops between strands of the blade and making contacts with conserved residues in the Ketch repeat. Furthermore, we identify a conserved C-H…π hydrogen bond between two key residues in the consensus Ketch repeat. This analysis extends our understanding of the structural roles of conserved residues in the Ketch repeat and highlights the potential rote of solvent in maintaining the fold of this common eukaryotic structural motif. [ABSTRACT FROM AUTHOR]

The X-ray structure of the enzyme 5-aminolaevulinic acid dehydratase (ALAD) from yeast complexed with the competitive inhibitor 5-hydroxylaevulinic acid has been determined at a resolution of 1.9 Å. The structure shows that the inhibitor is bound by a Schiff-base link to one of the invariant active-site lysine residues (Lys263). The inhibitor appears to bind in two well defined conformations and the interactions made by it suggest that it is a very close analogue of the substrate 5-aminolaevulinic acid (ALA). [ABSTRACT FROM AUTHOR]

Since the introduction of structural genomics, the protein has been recognized as the most important variable in crystallization. Recent strategies to modify a protein to improve crystal quality have included rationally engineered point mutations, truncations, deletions and fusions. Five naturally occurring variants, differing in 1-18 amino acids, of the 177-residue lectin domain of the F17G fimbrial adhesin were expressed and purified in identical ways. For four out of the five variants crystals were obtained, mostly in nonisomorphous space groups, with diffraction limits ranging between 2.4 and 1.1 Å resolution. A comparative analysis of the crystal-packing contacts revealed that the variable amino acids are often involved in lattice contacts and a single aminoacid substitution can suffice to radically change crystal packing. A statistical approach proved reliable to estimate the compatibilities of the variant sequences with the observed crystal forms. In conclusion, natural variation, universally present within prokaryotic species, is a valuable genetic resource that can be favourably employed to enhance the crystallization success rate with considerably less effort than other strategies. [ABSTRACT FROM AUTHOR]

Structural data are reported to 2.5 Å resolution for the first full analysis of the methotrexate-resistant Leu22Arg (L22R) variant of mouse dihydrofolate reductase (mDHFR) crystallized as a ternary complex with methotrexate (MTX) and the cofactor NADPH. These results are compared with the MTX and NADPH ternary complexes of L22R human DHFR (hDHFR) and those of mouse and human wild-type DHFR enzymes. The conformation of mDHFR Arg22 is such that it makes hydrogen-bonding contacts with Asp21, Trp24 and a structural water molecule, observations which were not made in the L22R hDHFR ternary complex. These data show that there is little difference between the structures of the wild type and L22R variant for either mouse or human DHFR; however, there are significant differences between the species. Comparison of these structures reveals that the active site of mDHFR is larger than that in the hDHFR structure. In mDHFR, the position of MTX is shifted 0.6 Å toward helix C (residues 59 ± 65), which in turn is shifted 1.2 Å away from the active site relative to that observed in the hDHFR ternary complexes. In the L22R variant mDHFR structure, MTX makes shorter contacts to the conserved residues Ile7, Val115 and Tyr121 than in the L22R variant human DHFR structure. These contacts are comparable in both wild-type enzymes. The unexpected results from this comparison of the mouse and human DHFR complexes bound with the same ligand and cofactor illustrate the importance of detailed study of several species of enzyme, even when there is a high sequence homology between them. These data suggest that the differences in binding interactions of the L22R variant are in agreement with the weaker binding affinity for MTX in the variant enzymes; the larger size of the binding site in mDHFR supports the observation that the binding affinity of MTX for L22R mDHFR is significantly weaker than that of the L22R hDHFR enzyme. [ABSTRACT FROM AUTHOR]

Ferredoxin-NADP(H) reductase (272 amino acids) from Rhodobacter capsulatus (FPR) has recently been postulated to be involved in the antioxidant response and to facilitate the provision of reduced flavodoxin for the reduction of nitrogenase. Crystallization trials of recombinant FPR were carried out at 291 K by the hanging-drop vapour-diffusion method. Orthorhombic crystals (unit-cell parameters a = 69.3, b = 93.6, c = 103.5 Å) were obtained. However, their diffraction pattern was not satisfactory and an extensive detergent screening was carried out over the initial crystallization conditions. The introduction of n-heptyl-β-D-thioglucoside produced new trigonal crystals (space group P3121; unit-cell parameters a = b = 120.5, c = 51.1 Å) that diffracted to 1.8 Å resolution at beamline BM16 at the ESRF. Preliminary structural analysis indicated that detergent molecules could increase the quality of diffraction of FPR crystals by stabilizing the disordered regions of the protein and by increasing the number of contacts in the crystal packing. [ABSTRACT FROM AUTHOR]

One of the most important aspects of macromolecular structure refinement is the use of prior chemical knowledge. Bond lengths, bond angles and other chemical properties are used in restrained refinement as subsidiary conditions. This contribution describes the organization and some aspects of the use of the flexible and human/machine-readable dictionary of prior chemical knowledge used by the maximum-likelihood macromolecular-refinement program REFMAC5. The dictionary stores information about monomers which represent the constitutive building blocks of biological macro- molecules (amino acids, nucleic acids and saccharides) and about numerous organic/inorganic compounds commonly found in macromolecular crystallography. It also describes the modifications the building blocks undergo as a result of chemical reactions and the links required for polymer formation. More than 2000 monomer entries, 100 modification entries and 200 link entries are currently available. Algorithms and tools for updating and adding new entries to the dictionary have also been developed and are presented here. in many cases, the REFMAC5 dictionary allows entirely automatic generation of restraints within REFMAC5 refinement runs. [ABSTRACT FROM AUTHOR]

Substructure-solution and phasing procedures using a combination of anomalous scattering and radiation-damage-induced isomorphous differences have been investigated. The tyrosine residues in thaumatin were iodinated with N-iodosuccinimide in the crystalline form as well as prior to crystallization. Several data sets were collected from both forms and used for substructure solution and phasing using various protocols, employing anomalous, isomorphous or both these signals. It was shown that combination of the anomalous and isomorphous signals in the form of the RIPAS (radiation-damage-induced phasing with anomalous scattering) strategy is beneficial for both locating the substructure and subsequent phasing. [ABSTRACT FROM AUTHOR]

Structural data are reported for SSP-19, a sperm-specific protein (SSP) family member from Caenorhabditis elegans. The SSP family [also known as the major sperm protein-like (MSP-like) family] contains proteins with only 107–109 amino acids, compared with 127 amino acids in the major sperm protein (MSP) family. MSP, the most abundant protein in nematode sperm, forms a dynamic actin-like cytoskeleton that provides the framework for the nematode sperm motility. In vivo, MSP dimers polymerize to form filaments that are constructed from two helical strands, which assemble into larger macromolecular structures. Little is known about the SSP family and a similar function is inferred from sequence and structural homology [Pfam (Protein Families Database of Alignments and HMMs) and SCOP (Structural Classification of Proteins) classification]. Despite the overall structural homology, the monomer-monomer interactions in SSP-19 are strikingly different from the interactions in the two MSP canonic domains described previously. [ABSTRACT FROM AUTHOR]

Serine acetyltransferase (SAT), is an enzyme that has been shown to catalyze the conversion of acetyl-CoA and L-serine to CoA and O-acetyl- L-serine in microorganisms and higher plants. SAT is a member of a family of hexapeptide- containing transferases. All dimer-of-trimer interactions are mediated through amino acids within an N-terminal extension common only to a subset of SAT, suggesting that members of this subfamily may also adopt hexameric structures. Comparisons of the Haemophilus influenzae SAT structure with related enzymes suggest by analogy that the active sites in Haemophilus influenzae SAT are likely to be formed by a crevice between adjacent threefold-related protomers.

The article discusses Ab initio phasing based on topological restraints. The phasing procedure consists of several steps. A large number of trial phase sets are generated randomly. For each of these phase sets, a Fourier synthesis of a specified resolution is calculated and a binary mask corresponding to the region of the highest synthesis values is defined in accordance with the specified cut-off level. The trial phase sets are considered as admissible and selected for further treatment if the connectivity-analysis output list satisfies the selection rules, formulated in terms of the total number of components, the number of compounds having the same volume, their finiteness and so on.

Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 Å apo PTP1B structure has been obtained, showing four highly coordinated water molecules in the active-site pocket of the enzyme; hence, the active site is highly solvated in the apo state. Three of the water molecules are located at positions that approximately correspond to the positions of the phosphate O atoms of the natural substrate phosphotyrosine and form a similar network of hydrogen bonds. The active-site WPD-Ioop was found to be in the closed conformation, in contrast to previous observations of wild- type PTPs in the apo state, in which the WPD-Ioop is open. The closed conformation is stabilized by a network of hydrogen bonds. These results provide new insights into and understanding of the active site of PTP1B and form a novel basis for structure-based inhibitor design. [ABSTRACT FROM AUTHOR]

The reversible dephosphorylation of β-aspartyl phosphate to L-aspartate-β-semialdehyde (ASA) in the aspartate bio-synthetic pathway is catalyzed by aspartate-β-semialdehyde dehydrogenase (ASADH). The product of this reaction is a key intermediate in the biosynthesis of diaminopimelic acid, an integral component of bacterial cell walls and a metabolic precursor of lysine and also a precursor in the biosynthesis of threonine, isoleucine and methionine. The structures of selected Haemophilus influenzae ASADH mutants were determined in order to evaluate the residues that are proposed to interact with the substrates ASA or phosphate. The substrate Km values are not altered by replacement of either an active-site arginine (Arg270) with a lysine or a putative phosphate-binding group (Lys246) with an arginine. However, the interaction of phosphate with the enzyme is adversely affected by replacement of Arg103 with lysine and is significantly altered when a neutral leucine is substituted at this position. A conservative Glu243 to aspartate mutant does not alter either ASA or phosphate binding, but instead results in an eightfold increase in the Km for the coenzyme NADP. Each of the mutations is shown to cause specific subtle active-site structural alterations and each of these changes results in decreases in catalytic efficiency ranging from significant (∼ 3% native activity) to substantial (<0.1% native activity). [ABSTRACT FROM AUTHOR]

Ornithine cyclodeaminase (OCD) is a member of the μ-crystallin protein family, the biological activity of which is the conversion of L-ornithine to L-proline and ammonia. In order to elucidate the functional groups of this enzyme that arc involved in catalysis. the crystallization of OCL) from Pseudomonas Putida was undertaken. Using microbatch-under-oil screening at the high-throughput crystallization laboratory (HTC) at the Hauptman-Woodward Medical Research Institute Inc. (HWI Buffalo, NY, USA), numerous crystallization conditions were rapidly identified. Several conditions could he reproduced on a larger scale as vapor-diffusion experiments in-house. The best diffraction-quality crystals were obtained front solutions of 40%(v/v) 2-methyl-2,4-pentanediol buffered at pH 6.0 with 0.1 M MES and diffracted X-rays to 1.68 Å resolution. Crystals belonged to space group P212121, with unit-cell parameters a = 70.0, b = 78.3, c = 119.4 Å. The VM was 2.1 ų Da-1. corresponding to 42% solvent, which is consistent with two 38.5 kDa molecules per asymmetric unit. The structure determination is under way using experimental phasing methods. [ABSTRACT FROM AUTHOR]

A set of tables is presented and a survey given of the architecture of metal coordination groups in a representative set of protein structures from the Protein Data Bank [Bernstein et al. (1977). J. Mol. Biol. 112. 535-542: Berman et al. (2000). Nucleic Acids Ret 28. 235-242]. The structures have been determined to a resolution of 2.5 Å or better: the metals considered are Ca. Mg. Mn. Fe. Cu. Zn. Na and K. with particular emphasis on Ca and Zn and the exclusion of haem groups and Fe/S clusters: the proteins are a representative set in which none has more than 30% sequence identity with any other. In them the metal is coordinated by several donor groups from different amino-acid residues in the protein chain and often also by water or other small molecules. The tables. ∼600 metal coordination groups. include information on the conformations of the protein chain in the region around the metal and reliability indicators. They illustrate the wide variety of coordination numbers. chelate-loop sizes and other properties and the different characteristics of different metals. They show that glycine has a particular significance in the position adjacent to a donor residue, especially in Ca coordination groups. They also show that metal coordination does not appear to lead to significant distortions of the torsion angles ϕ, ψ from their normally allowed values. Very few metal coordination groups occur more than once in the representative set and when they do they are usually related in fold and function: they have similar but not necessarily identical conformations. However, individual chelate loops, for example Zn(-C-X-X'-C-), in which both cysteines are coordinated to Zn through S. and X and X' are any amino acids, are repeated frequently in many different and unrelated proteins. Not all chelate loops with the same composition have the same conformation, but for smaller loops there are usually one or two strongly preferred and well defined conformations. Quite frequently more than one metal coordination group is associated with one protein chain: these proteins are identified. [ABSTRACT FROM AUTHOR]

Investigates the impact of lysine to arginine surface mutations on the crystallization of the globular domain of RhoGDI. Data showing that lysine-arginine mutants are more likely to crystallize than the wild-type protein; Major crystal contacts in the new lattice created by the mutated epitope.

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the condensalion of tive isopentenyl pyrophosphates with farnesyl pyrophosphate to generate C[SUB40] octaprenyl pyrophosphate. The enzymes from the hyperthermophilic bacterium Thermotoga maritima and from the mesophilic Escherichia coli were expressed in E. coli and the recombinant proteins were purified and crystallized. The T. maritima OPPs crystals belong to space group P42[SUB1]2. with unit-cell parameters a = b = 151.53. c = 69.72 &Aring. The E. coli OPPs crystals belong to space group C222[SUB1], with unit-cell parameters a = 247.66. b = 266.10. c = 157.93 Å. Diffraction data were collected at 100 K using synchrotron radiation and an in-house X-ray source. Structure determination of T. maritima OPPs has been carried out using MIR data sets at 2.8 Å resolution. The asymmetric unit contains one dimer. An initial model with 280 residues per subunit has been built and refined to 2.28 Å resolution. It shows mostly helical structure and resembles that of avian farnesyl pyrophosphate synthase. [ABSTRACT FROM AUTHOR]

The structure elucidation of the α-amino acid ester hydrolase from Acetobacter turbidans by molecular replacement is described. In the monoclinic crystal, the molecules are related by both rotational and pseudo-crystallographic translational NCS (non-crystallographic symmetry). Refinement of the structure converged at unacceptably high R factors. After reevaluation of the data. it was found that the crystal was merohedrally twinned, with a high twinning fraction. It is shown that the pseudo-crystallographic NCS causes aberrant behaviour of conventional twinning indicators, which explains why the twinning was only realized at the refinement stage. [ABSTRACT FROM AUTHOR]

The crystal structure of viscotoxin A3 (VT A3) extracted from European mistletoe (Viscum album L.) has been solved using the anomalous diffraction of the native S atoms measured in-house with Cu Kα radiation to a resolution of 2.2 Å and truncated to 2.5 Å. A 1.75 &Aalpha; resolution synchrotron data set was used for phase expansion and refinement. An innovation in the dual-space substructure-solution program SHELXD enabled the individual S atoms of the disulfide bonds to he located using the Cu Kα data: this resulted in a marked improvement in the phasing compared with the use of super-S atoms. The VT A3 monomer consists of 46 amino acids with three disulfide bridges and has an overall fold resembling the canonical architecture of the α- and β-thionins, a capital letter L. The asymmetric unit consists of two monomers related by a local twofold axis and held together by hydrophobic inter-actions between the monomer units. One phosphate anion (confirmed by [SUP31]P-NMR and MS) is associated with each monomer. [ABSTRACT FROM AUTHOR]

An algorithm for the automated macromolecular model building of polypeptide backbones is described. The procedure is hierarchical. In the initial stages, many overlapping polypeptide fragments are built. In subsequent stages, the fragments are extended and then connected. Identification of the locations of helical and β-strand regions is carried out by FFT-based template matching. Fragment libraries of helices and β-strands from refined protein structures are then positioned at the potential locations of helices and strands and the longest segments that fit the electron-density map are chosen. The helices and strands are then extended using fragment libraries consisting of sequences three amino acids long derived from refined protein structures. The resulting segments of polypeptide chain are then connected by choosing those which overlap at two or more C[sup α] positions. The fully automated procedure has been implemented in RESOLVE and is capable of model building at resolutions as low as 3.5 Å. The algorithm is useful for building a preliminary main-chain model that can serve as a basis for refinement and side-chain addition. [ABSTRACT FROM AUTHOR]

Leucine dehydrogenase is an octameric enzyme which belongs to the superfamily of amino-acid dehydrogenases and catalyses the reversible oxidative deamination of leucine to 2-ketoisocaproate, with the corresponding reduction of the cofactor NAD+. Catalysis by this enzyme is thought to involve a large-scale motion of the enzyme's two domains between an 'open' and 'closed' form, with the latter representing a conformation of the enzyme in which the partners involved in the hydride-transfer reaction are appropriately positioned for catalysis. Whilst a structure for the open form of the enzyme has been determined, the nature of the closed form has yet to be observed. In order to trap a closed form, crystals of the complexes of leucine dehydrogenase from Thermoactinomyces intermedius with 2-ketoisocaproate and with 2-ketoisocaproate and NAD+ have been obtained by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals of the binary complex with 2-ketoisocaproate belong to space group P212121, with approximate unit-cell parameters a = 106, b = 118, c = 320 Šand an octamer in the asymmetric unit, corresponding to a VM of 3.1 ų Da-1. The crystals of the non-productive ternary complex belong to space group P61 or P65, with approximate unit-cell parameters a = b = 117, c = 502 Šand an octamer in the asymmetric unit, corresponding to a VM of 3.0 A3 Da-1. These crystals diffract X-rays on a synchrotron- radiation source to at least 2.8 and 3.3 Šresolution, respectively, and are suitable for a full structure determination. [ABSTRACT FROM AUTHOR]

The main-chain conformations of 237 384 amino acids in 1042 protein subunits from the PDB were analyzed with Ramachandran plots. The populated areas of the empirical Ramachandran plot differed markedly from the classical plot in all regions. All amino acids in α-helices are found within a very narrow range of φ, ψ angles. As many as 40% of all amino acids are found in this most populated region, covering only 2% of the Ramachandran plot. The β-sheet region is clearly subdivided into two distinct regions. These do not arise from the parallel and antiparallel β-strands, which have quite similar conformations. One β region is mainly from amino acids in random coil. The third and smallest populated area of the Ramachandran plot, often denoted left-handed α-helix, has a different position than that originally suggested by Ramachandran. Each of the 20 amino acids has its own very characteristic Ramachandran plot. Most of the glycines have conformations that were considered to be less favoured. These results may be useful for checking secondary-structure assignments in the PDB and for predicting protein folding. [ABSTRACT FROM AUTHOR]

Cytotoxic necrotizing factor 1 (CNF1), a 114 kDa toxin produced by certain pathogenic strains of Escherichia coli, constitutively activates members of the Rho GTPase family, leading to cytopathic effects. The toxin inhibits GTP turnover by Rho proteins through site-specific deamidation of a Rho glutamine required for GTP hydrolysis. To understand the basis for catalytic activity and target specificity of CNF1, the structure of a catalytically active fragment of CNF1 was sought. Here, strategies that led to successful expression of a soluble 33 kDa active fragment, growth and improvement in the quality of the crystals and determination of phases using a quadruple methionine-substitution mutant of the fragment are presented. [ABSTRACT FROM AUTHOR]

The greylag goose (Anser anser), which lives on lowlands and cannot tolerate hypoxic conditions, presents a striking contrast to its close relative the bar-headed goose (A. indicus), which lives at high altitude and possesses high-altitude hypoxia adaptation. There are only four amino-acid residue differences at α18, α63, α119 and β125 between the haemoglobins of the two species. The crystal structure of greylag goose oxy haemoglobin was determined at 3.09 Å resolution. Its quaternary structure is slightly different from that of the bar-headed goose oxy haemoglobin, with a rotation of 2.8° in relative orientation of the two dimers. Of the four mutations, those at α119 and β125 produce contact changes in the α1β1 interlace and may be responsible for the differences in intrinsic oxygen affinity between the two species; those at α18 and α63 may be responsible for the differences in quaternary structure between the two species. [ABSTRACT FROM AUTHOR]

Polydocanol has a wide range of medical applications, especially in sclerotherapy of many diseases such as gastrointestinal antiplastia, oesophageal haemangioma etc. it is of interest to study the mode of binding of this medically important detergent and its subsequent action on proteins. Here, three crystal structures of serine protease trypsin are reported in the presence of varying concentrations of polydocanol in order to elucidate its mode of binding and interactions with proteins. Polydocanol binds to the protein with its hydrophilic head rather than the hydrophobic tail as is the case with other detergents such as SDS and MEGA-8. This hydrophilic binding mode results in the binding sites of polydocanol being distributed on the surface of the enzyme. There are at least 11 binding sites for polydocanol in trypsin. Polydocanol forms part of the large-scale water networks which connect distant regions of the enzyme, thereby stabilizing it. The hydrophilic binding of polydocanol also results in cross-linked pairs of tryp sin molecules. [ABSTRACT FROM AUTHOR]

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyses the decarboxylation of orotidine 5'-monophosphate to uridine 5'-monophosphate, the last step in the de novo biosynthesis of uridine 5'-monophosphate. In order to determine the structure of ODCase from Escherichia coli by the multi-wavelength anomalous dispersion technique, both native and SeMet-substituted proteins have been produced and purified. During the production of SeMet ODCase, it was observed that SeMet was the only amino acid that it was necessary to add to the defined medium during expression. SeMet-substituted ODCase in complex with the inhibitor 1-(5'-phospho-β-D-ribofuranosyl)barbituric acid crystallizes under similar conditions as the native enzyme. In contrast to the native enzyme, where the crystals belong to the orthorhombic space group P212121, the SeMet-substituted enzyme crystallizes in the monoclinic space group P21, with a quadrupling of the volume of the asymmetric unit. Despite the drastic difference in symmetry, the overall crystal packing is effectively identical in the two crystal forms. The change in space group appears to originate in differences in the crystal contacts near the SeMet and Met residues. These differences can be rationalized in terms of SeMet's larger size and hydrophobicity. [ABSTRACT FROM AUTHOR]

The structure of the Haemophilus influenzae HslU protein, a molecular chaperone of the Clp/Hsp100 family, has been solved to 2.3 Å by molecular replacement using a model of the homologous Escherichia coli protein. The crystals in which the structure was solved have an unusual twinning, or one-dimensional disorder, in which each successive crystal-packing layer is displaced laterally relative to the one below it. A model for the twinning and an algorithm for detwinning the data are described, it is known from other work that when the HslU hexamer binds its cognate protease HslV, the carboxy-terminal helices of HslU protomers distend and bind between HslV subunits. Comparison of HslU alone with its structure in the HslUV complex reveals several conserved amino-acid residues whose side-chain interactions differ between the two structures, suggesting that they may be part of a conformational switch that facilitates the release of the HslU carboxy-terminal helices when HslV binds. [ABSTRACT FROM AUTHOR]

The first observation of the unique environment for thyroxine (T4) binding in tetrameric rat transthyretin (rTTR) is reported as determined by X-ray diffraction. These data revealed different modes of hormone binding in the two unique hormone-binding sites in the rat TTR tetramer channel. Differences in the orientation of thyroxine and the position of water molecules in the two binding sites further suggest a mechanism for the docking pathway of the hormone into the channel of TTR. Crystals of the rat transthyretin-thyroxine complex are isomorphous with those reported for apo rTTR and crystallized in the tetragonal space group P43212 with four independent TTR monomeric subunits in the asymmetric part of the crystal lattice. Data were collected to 2.5 Å resolution and the structure was refined to R = 20.9% for 15 384 data in the resolution range 12-2.5 Å. Similar to human TTR, the rat protein is also a 54 000 Da tetramer with four identical polypeptide chains of 127 amino-acid residues. Of the 22 amino-acid residues which differ between the human and rat sequences, none are in the thyroxine-binding domains. Analysis of these structural data reveals that the tertiary structure is similar to that of hTTR, with only small differences in the flexible loop regions on the surface of the structure. Conformational changes of the amino acids in the channel result in a hydrogen-bonded network that connects the two binding domains, in contrast to the hydrogen bonds formed along the tetramer interface in the apo transthyretin structure. These changes suggest a mechanism for the signal transmission between thyroxine-binding domains. [ABSTRACT FROM AUTHOR]

The classical approach to building the amino-acid residues into the initial electron-density map requires days to weeks of a skilled investigator's time. Automating this procedure should not only save time, but has the potential to provide a more accurate starting model for input to refinement programs. The new software routine MAID builds the protein structure into the electron-density map in a series of sequential steps. The first step is the fitting of the secondary α-helix and, β-sheet structures. These `fits' are then used to determine the local amino-acid sequence assignment. These assigned fits are then extended through the loop regions and fused with the neighboring sheet or helix. The program was tested on the unaveraged 2.5 Å selenomethionine multiple-wavelength anomalous dispersion (SMAD) electron-density map that was originally used to solve the structure of the 291-residue protein human heart short-chain L-3-hydroxyacyl-COA dehydrogenase (SHAD). Inputting just the map density and the amino-acid sequence, MAID fitted 80% of the residues with an r.m.s.d. error of 0.43 Å for the main-chain atoms and 1.0 Å for all atoms without any user intervention. When tested on a higher quality 1.9 Å SMAD map, MAID correctly fitted 100% (418) of the residues. A major advantage of the MAID fitting procedure is that it maintains ideal bond lengths and angles and constrains ψ/&psi angles to the appropriate Ramachandran regions. Recycling the output of this new routine through a partial structure-refinement program may have the potential to completely automate the fitting of electron-density maps. [ABSTRACT FROM AUTHOR]

Rubidium is a monovalent metal that can be used as a counterion in protein solutions. X-ray anomalous scattering from rubidium ions bound to the protein surface was used for phasing of the crystal structure of the hsp60 apical domain from Thermus thermophilus. Multiple-wavelength anomalous dispersion (MAD) data were collected from a crystal obtained from a solution containing 0.2 M rubidium salt. One molecule of protein (147 amino acids) binds one well ordered and one poorly ordered Rb atom. Phases calculated with the program SHARP were sufficient for automatic tracing and side-chain assignment using the program ARP/wARP. The data show that bound rubidium ions can be used to determine protein structures and to study the interaction of monovalent metal ions with proteins and other macromolecules. [ABSTRACT FROM AUTHOR]

The crystallization conditions of a recombinant form of the complete sequence of human γ-interferon, designated r-hu IFN-γ (RU 42369), have been determined after studying the behaviour of this protein in solution by small-angle X-ray scattering (SAXS) as a function of pH and salt type. IFN-γ is difficult to crystallize without truncating at least the last five amino acids of the C-terminus; the SAXS results suggest viable crystallization conditions that led to crystals of r-hu IFN-γ suitable for X-ray diffraction analysis. The crystals were grown in the presence of ammonium sulfate using vapour-diffusion techniques. The crystals, which diffract to 5 Å resolution at best, belong to the primitive tetragonal space group P4212 and have unit-cell parameters a = b = 123.4, c = 93.4 Å. The protein contained in these crystals was analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), which verified the presence of the complete amino-acid sequence of r-hu IFN-γ. [ABSTRACT FROM AUTHOR]

Haemoglobin from the bar-headed goose (Anser indicus) has higher oxygen affinity than that from its lowland relatives such as greylag goose (A. anser). The crystal structure of bar-headed goose aquomet haemoglobin was determined at 2.3 A resolution and compared with the structures of the goose oxy, human, horse and other avian haemoglobins and the sequences of other avian haemoglobins. Four amino-acid residues differ between greylag goose and bar-headed goose haemoglobins, among which Alaα119 and Aspβ125 in bar-headed goose haemoglobin reduces the contacts between the α1 and β1 subunits compared with Pro and Glu, respectively, and therefore may increase the oxygen affinity by loosening the α1β1 interlace. Compared with human oxy haemoglobin, the relative orientation of two αβ dimers in the bar-headed goose aquomet and oxy Hbs are rotated by about 4°, indicating a unique quaternary structural difference from the typical R state. This new `RH' state is probably correlated with the higher oxygen affinity of bar-headed goose haemoglobin. [ABSTRACT FROM AUTHOR]

Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A. Truncation of seven amino-acid residues from the amino-terminal sequence resulted in RNase 1ΔN7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI). This RNase I variant has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data to 1.9 Å resolution. The molecule displays the α + β folding topology typical of members of the RNase A superfamily. The main distinct features found in RNase 1ΔAN7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite. These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1ΔN7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme. [ABSTRACT FROM AUTHOR]

The article presents information on the structure of human a-thrombin complexed with RWJ-51438 at 1.7 Å an unusual perturbation of the 60A--60I insertion loop. The ketone carbonyl group of the inhibitor is covalently linked to the hydroxyl O atom of Ser195, forming a tetrahedral intermediate hemiketal structure; the benzothiazole ring N atom of RWJ-51438 forms a hydrogen bond with His57. The amino group of the Lys60F does not form a hydrogen bond with the carbonyl O atom of His57.

A protein sequence can be classified into one of four structural classes, namely α, β, α + β and α/β, based on its amino-acid composition. The present study aims at understanding why a particular sequence with a given amino-acid composition should fold into a specific structural class. In order to answer this question, each amino acid in the protein sequence was classified to a particular neighbor density based on the number of spatial residues surrounding it within a distance of 6.5 Å. Each of the four structural classes showed a unique preference of amino acids in each of the neighbor densities. Residues which show a high compositional bias in a structural class are also found to occur in high neighbor densities. This high compositional bias towards specific residues in the four different structural classes of proteins appears to be caused by structural and functional requirements. The distribution of amino acids in different neighbor densities is graphically presented in a novel logo form which incorporates several features such as composition, the frequency of occurrence and color code for amino acids. The spatial neighbors of the residues in different neighbor densities and their secondary structural location are also represented in the form of logos. This representation helped in the identification of specific details of the whole data which may otherwise have gone unnoticed. It is suggested that the data presented in this study may be useful in knowledge-based structure modelling and de novo protein design. [ABSTRACT FROM AUTHOR]

The structure of canavalin was refined to 2.1 and 2.0 Å resolution in cubic and hexagonal crystals of space group P213 and P63, respectively. The threefold molecular symmetry is expressed in the symmetry of both crystals, where each identical subunit is an asymmetric unit. The canavalin subunit consists of two very similar domains, each comprised of a core subdomain having Swiss-roll topology with a loop subdomain that contains helices. The refined canavalin models resolved the discrepancy in amino-acid registers of the secondary- structural elements compared with phaseolin. The presence of strand Z in both domains of canavalin was confirmed and a new helix in the ioop between strands A and B of each domain was observed. The models were analyzed in terms of the duplicated vicilin domains. Three strictly conserved residues, two glycines and a proline, were identified. The similarity between entire vicilin molecules is greater than that between separate domains of canavalin and phaseolin. Homology modeling of the sucrose-binding protein (SBP) from soybean showed a plausible trimeric assembly of subunits similar to that of vicilins. [ABSTRACT FROM AUTHOR]

Regucalcin is a novel calcium ion (Ca2+) binding protein that does not contain an EF-hand motif as a Ca2+-binding domain and has been demonstrated to play a multi-functional role in many cell types. Human liver regucalcin, consisting of 299 amino-acid residues, was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method in the presence of polyethylene glycol 4000 as a precipitant. A native crystal diffracted to 2.8 Å with synchrotron radiation and belongs to space group P21, with unit-cell parameters a = 64.87, b = 52.52, c = 86.38 Å, β = 99.86°. Two molecules most probably exist in the asymmetric unit, corresponding to VM 2.2 ų Da-1. Heavy-atom derivative data were collected and the Pb derivative showed one high-occupancy site per molecule. [ABSTRACT FROM AUTHOR]

The CP1 domain (the editing domain) of isoleucyl-tRNA synthetase (IleRS) hydrolyzes misactivated Val-AMP in pre-transfer editing and mischarged Val-tRNAIle in post-transfer editing. The CP1 domain of Thermus thermophilus IleRS was expressed in isolation, purified and cocrystallized with Val-AMS (a Val-AMP analogue) and with Val-2AA (a Val-tRNAIle analogue). Two different expression constructs were used for each cocrystallization. The complex crystals with Val-AMS belong to the tetragonal space group P41212, with unit-cell parameters a = b = 102.00, c = 84.88 Å. The asymmetric unit contains two molecules of the CP1 domain, with a corresponding crystal volume per protein weight of 2.8Å3 Da-1 and a solvent content of 53.5%. The complex crystals with Val-2AA belong to the tetragonal space group P4122, with unit-cell parameters a = b = 72.59, c= 83.68 Å. The asymmetric unit contains one molecule of the CP1 domain, with a corresponding crystal volume per protein weight of 2.8 A 3 Da-1 and a solvent content of 55.8%. Data sets diffracting to 1.7 Å resolution were collected from each single crystal at 100 K. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases establish the rules of the genetic code by attaching the correct amino acid to their cognate-tRNA. Recent structural and biochemical work on both classes of enzymes has provided valuable insights into how very closely related amino-acid substrates are discriminated with very high accuracy in order to maintain high fidelity during translation of the genetic code. Threonyl-tRNA synthetase is a class II enzyme and faces a crucial double-discrimination problem of selecting threonine from valine and serine.

The adrenal gland protein AD-004 was identified in the human adrenal gland. Full-length AD-004 contains 172 amino acids, with a predicted molecular weight of about 20 kDa. In attempts to crystallize human AD-004, the gene was subcloned into a modified pET vector, pET21-DEST, with an N-terminal His5 tag using the Gateway cloning system, followed by protein expression in Escherichia coli strain BL21(DE3). The protein was purified in two steps to near-homogeneity and was crystallized. The crystals belong to space group P61 or P65, with unit-cell parameters a = b = 99.56, c = 57.19 Å. A complete 2.0 Å data set has been collected at a rotating-anode X-ray source and structure determination is under way. [ABSTRACT FROM AUTHOR]

α-Isopropylmalate synthase catalyses the aldol condensation of α-ketoisovalerate and acetyl coenzyme A to produce α-isopropylmalate. This reaction is the first committed step of leucine biosynthesis, which is interrelated with the pathways for production of the other branched-chain amino acids, valine and isoleucine. The absence of these pathways in mammals suggests that these enzymes could be useful targets for drug design against microbial pathogens. The gene for α-IPMS in Mycobacterium tuberculosis (Rv3710) has been cloned, expressed in Escherichia coli, both in native and selenomethionine-substituted forms, and crystallized. The SeMet crystals are suitable for high-resolution X-ray structural analysis. These crystals are monoclinic, with unit-cell parameters a = 54.25, b = 154.73, c = 68.82 Å, space group P21 and two molecules in the asymmetric unit. X-ray diffraction data to 2.0 Å resolution have been collected. [ABSTRACT FROM AUTHOR]

PDZ-containing RhoGEF (PDZ-RhoGEF) is a multidomain protein composed of 1522 amino acids that belongs to the guanine nucleotide exchange factors family (GEF) active on Rho GTPases. It is highly specific for RhoA and is thought to transduce signals from Gα12/13-coupled receptors ot the RhoA-dependent regulatory cascades. The protein shows high sequence homology to LARG, p115-RhoGEF and Drosophila DRhoGEF2. The exchange reaction is catalyzed by a DH domain, which is directly downstream of a PH domain in all known Rho-specific GEFs. The DH/PH tandem of PDZ-RhoGEF and C-terminally truncated RhoA were expressed in Escherichia coli as TEV protease-cleavable fusion proteins containing GST and a hexahistidine tag at the N-termini, respectively. The nucleotide-free DH/PH RhoA complex was purified by gel filtration and crystallized. The Crystals belong to space group P21, with unit-cell parameters a = 88.6, b = 119.0, c = 91.5 Å, β = 114.7. [ABSTRACT FROM AUTHOR]

IBI is a mammalian scaffold protein that interacts with components of the c-Jun N-terminal kinase (JNK) signal-transduction pathway mainly via its protein-protein interaction domains. Crystallization of the key Src homology 3 (SH3) domain of IBI has been achieved. Crystallization experiments with unmodified protein and deliberately oxidized protein have led to different crystal forms. X-ray data have been collected to 3.0 Å resolution from a crystal form with rectangular prism morphology. These crystals are orthorhombic (P2[SUB1]2[SUB1]2[SUB1], with unit-cell parameters a = 45.9, b = 57.0. c = 145.5 A. These are the first crystallographic data on a scaffold molecule, such as IBI to be reported. [ABSTRACT FROM AUTHOR]

FKBP52 is a high-molecular-weight immunophilin belonging to the FKBP (FK506-binding protein) family. FKBP52 is one of several chaperome proteins associated with untransformed steroid receptors in steroid receptor-hsp90 heterocomplexes. Here, the C-terminal domain (amino acids 145 459) has been cloned, overexpressed and purified. Crystals were obtained using the hanging-drop vapourdiffusion technique with ammonium sulfate as precipitant in 0.1 M Tris pH 8.0 solution. Diffraction data to 2.7 Å were collected from a selenomethionine-containing crystal belonging to space group C222[SUB1]. with unit-cell parameters u = l 14.4.h = 143.1.c = 171.2 Å α = β = γ = 90. There are three molecules per asymmetric unit. [ABSTRACT FROM AUTHOR]

A small DNA-binding protein of 87 amino-acid residues from the hyperthermophilic archaeon Methanococcus jannaschii (Mja10b) was cloned and overexpressed in Escherichia coli. The protein was crystallized and the crystals belong to the space group P6122/P6522, with unit-cell parameters a = b = 50.85, c = 124.02 Å, α = β = 90, γ = 120°. The crystals diffracted to a maximum resolution of 2.2 Å at 100 K using Cu Kα radiation. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (VM) of 2.4 ų Da-1 and a solvent content of 49% by volume. A full set of X-ray diffraction data was collected to 2.2 Å from the native crystal. [ABSTRACT FROM AUTHOR]