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Not all proteins form well defined three-dimensional structures in their native states. Some amino-acid sequences appear to strongly favour the disordered state, whereas some can apparently transition between disordered and ordered states under the influence of changes in the biological environment, thereby playing an important role in processes such as signalling. Although important biologically, for the structural biologist disordered regions of proteins can be disastrous even preventing successful structure determination. The accurate prediction of disorder is therefore important, not least for directing the design of expression constructs so as to maximize the chances of successful structure determination. Such design criteria have become integral to the construct-design strategies of laboratories within the Structural Proteomics In Europe (SPINE) consortium. This paper assesses the current state of the art in disorder prediction in terms of prediction reliability and considers how best to use these methods to guide construct design. Finally, it presents a brief discussion as to how methods of prediction might be improved in the future. [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]

The structure of sylvaticin, a 10 kDa major pythin protein excreted by the parasitic oomycete Pythium sylvaticum, has been determined. Although closely related to α-elicitins in its biological response, toxicity and overall structure, sylvaticin presents a number of structural features that make it an unusual member of the elicitin class. Elicitins possess a large hydrophobic cavity and the mechanism of the systemic acquired resistance induced in planta is known to proceed through lipid transport and complexation within this cavity. Unlike other elicitins, sylvaticin contains tryptophan residues, one of which points inwards towards the central cavity, thus limiting access to sterols. In the case of sylvaticin, the sterol-transport mechanism is likely to be of less importance compared with other members of the elicitin family and still remains to be fully characterized. [ABSTRACT FROM AUTHOR]

In the proteolytic pathway of prokaryotic and eukaryotic organisms, proteins tagged for proteolysis are firstly shredded into smaller peptides by compartmentalized proteases such as the proteasome complex. Accordingly, a variety of downstream proteases have evolved to further hydrolyze these peptides to the level of free amino acids. In the search for such downstream proteases, a high-molecular-weight protease complex called trilobed protease (TLP) was recently discovered in the archaeon Pyroccocus furiosus. The crystal structure of the N-terminal β-propeller domain of the trilobed protease at 2 Å resolution shows that the trilobed protease utilizes this accessory domain to control substrate access to the active site. Modelling of the intact TLP monomer suggests that this protease has an additional side entrance to its active site as in the DPP-IV or tricorn protease complexes. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatases (PTPs) play roles in many biological processes and are considered to be important targets for drug discovery. As inhibitor development has proven challenging, crystal structure-based design will be very helpful to advance inhibitor potency and selectivity. Successful application of protein crystallography to drug discovery heavily relies on high-quality crystal structures of the protein of interest complexed with pharmaceutically interesting ligands. It is very important to be able to produce protein–ligand crystals rapidly and reproducibly for as many ligands as necessary. This study details our efforts to engineer the catalytic domain of human protein tyrosine phosphatase β (HPTPβ-CD) with properties suitable for rapid-turnaround crystallography. Structures of apo HPTPβ-CD and its complexes with several novel small-molecule inhibitors are presented here for the first time. [ABSTRACT FROM AUTHOR]

The Bug proteins form a large family of periplasmic solute-binding receptors present in a number of bacterial species. Here, the crystal structure of Bordetella pertussis BugE, a member of the Bug family coded by the gene BP0250, is reported. It adopts the Venus flytrap architecture of periplasmic binding proteins, with two domains separated by a deep cleft. BugE has a bound ligand, identified as a glutamate. The structure of B. pertussis BugD, which is an aspartic acid transporter, has recently been reported. These structures reveal high conservation of the Bug architecture, despite limited sequence identity. They share a common carboxylate-binding motif defined by two strand–β-turn–α-­helix motifs, also involving two water molecules to bridge the carboxylate O atoms to the protein. The two water molecules are hydrogen bonded to a common main-chain carbonyl group. Although the features of the carboxylate-binding motif are totally conserved, the ligand in BugE is bound by its side-chain carboxylate group rather than by its α-­carboxylate as in BugD. This specific ligand-binding motif is highly conserved in Bug proteins and the BugE structure suggests that the cavity of the Bug proteins might also accommodate carboxylated solutes other than amino acids. The vast expansion of the Bug family in several bacterial genera is likely to be explained by the possible diversity of ligands. No charged residues are involved in glutamate binding by BugE, unlike what has been described for all glutamate receptors reported so far. [ABSTRACT FROM AUTHOR]

A database of invarioms for structural refinement of amino-acid, oligopeptide and protein molecules is presented. The spherical scattering factors of the independent atom or promolecule model are replaced by `individual' aspherical scattering factors that take into account the chemical environment of a bonded atom. All amino acids were analysed in terms of their invariom fragments. In order to generate 73 database entries that cover this class of compounds, 37 model compounds were geometry-optimized and theoretical structure factors were calculated. Multipole refinements were then performed on these theoretical structure factors to yield the invariom database. Validation of this database on an extensive number of experimental small-molecule crystal structures of varying quality and resolution shows that invariom modelling improves various figures of merit. Differences in figures of merit between invariom and promolecule models give insight into the importance of disorder for future protein-invariom refinements. The suitability of structural data for application of invarioms can be predicted by Cruickshank's diffraction-component precision index [Cruickshank (1999), Acta Cryst. D 55, 583–601]. [ABSTRACT FROM AUTHOR]

The Escherichia coli enterobactin synthetic cluster is composed of six proteins, EntA–EntF, that form the enterobactin molecule from three serine molecules and three molecules of 2,3-dihydroxybenzoic acid (DHB). EntC, EntB and EntA catalyze the three-step synthesis of DHB from chorismate. EntA is a member of the short-chain oxidoreductase (SCOR) family of proteins and catalyzes the final step in DHB synthesis, the NAD+-dependent oxidation of 2,3-­dihydro-2,3-dihydroxybenzoic acid to DHB. The structure of EntA has been determined by multi-wavelength anomalous dispersion methods. Here, the 2.0 Å crystal structure of EntA in the unliganded form is presented. Analysis of the structure in light of recent structural and bioinformatic analysis of other members of the SCOR family provides insight into the residues involved in cofactor and substrate binding. [ABSTRACT FROM AUTHOR]

The structure of the Rieske-type ferredoxin (T4moC) from toluene 4-monooxygenase was determined by X-ray crystallography in the [2Fe–2S]2+ state at a resolution of 1.48 Å using single-wavelength anomalous dispersion phasing with the [2Fe–2S] center. The structure consists of ten β-strands arranged into the three antiparallel β-sheet topology observed in all Rieske proteins. Trp69 of T4moC is adjacent to the [2Fe–­2S] centre, which displaces a loop containing the conserved Pro81 by ∼8 Å away from the [2Fe–2S] cluster compared with the Pro loop in the closest structural and functional homolog, the Rieske-type ferredoxin BphF from biphenyl dioxygenase. In addition, T4moC contains five hydrogen bonds to the [2Fe–2S] cluster compared with three hydrogen bonds in BphF. Moreover, the electrostatic surface of T4moC is distinct from that of BphF. These structural differences are identified as possible contributors to the evolutionary specialization of soluble Rieske-type ferredoxins between the diiron monooxygenases and cis-dihydrodiol-forming dioxygenases. [ABSTRACT FROM AUTHOR]

Pleckstrin is an important intracellular protein involved in the phosphoinositide-signalling pathways of platelet activation. This protein contains both N- and C-terminal pleckstrin-homology (PH) domains (N-PH and C-PH). The crystal structure of C-PH was solved by molecular replacement and refined at 2.1 Å resolution. Two molecules were observed within the asymmetric unit and it is proposed that the resulting dimer interface could contribute to the previously observed oligomerization of pleckstrin in resting platelets. Structural comparisons between the phosphoinositide-binding loops of the C-PH crystal structure and the PH domains of DAPP1 and TAPP1, the N-terminal PH domain of pleckstrin and a recently described solution structure of C-PH are presented and discussed. [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]

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 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 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 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]

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]

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]

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]

SEDL (known also as sedlin) is a 140 amino-acid protein with a putative role in endoplasmic reticulum-to-Golgi transport. Several missense mutations and deletion mutations in the SEDL gene, which result in protein truncation by frame shift, are responsible for spondyloepiphyseal dysplasia tarda, a progressive skeletal disorder. The protein is identical to MIP-2A, which was shown to interact physically with c-myc promotor-binding protein 1 (MBP-1) and relieve the regulatory role of MBP-1 as a general transcription repressor. In order to gain insights into the function of SEDL by structural analysis, the protein was overexpressed and crystallized as a first step. SEDL was overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method at 298 K. The crystals belong to the orthorhombic space group C2221, with unit-cell parameters a = 46.69, b = 101.30, c = 66.15 Å. The unit cell is likely to contain one molecule of SEDL, with a crystal volume per protein mass (VM) of 2.36 ų Da-1 and a solvent content of about 47.9% by volume. A native data set to 2.8 Å resolution was obtained from a flash-cooled crystal using synchrotron radiation. [ABSTRACT FROM AUTHOR]

A novel tripeptide motif called a nest has recently been described in proteins with the function of binding anionic, or partially anionic, atoms such as carbonyl 0 atoms. In the present work, a search for nests in small polypeptides stored in the Cambridge Structural Database is reported. 37 unique examples were found: over half form part of hydrogen-bond arrangements resembling those in proteins, such as Schellman/ paperclip loop motifs, various types of β-turn and Asx-turns or Ser/Thr-turns, while a third are in novel situations, some involving binding to anionic groups from other molecules within the crystal complex. An example is the antibiotic vancomycin, which incorporates a prominent nest forming part of a peptide-binding site. This nest binds the carboxylate of the C-terminal D-alanine of the bacterial cell-wall precursor peptide, thereby inhibiting the final step of bacterial cell-wall synthesis. As in proteins, a number of nests occur in short peptides with an alternating glycine/L-amino- acid sequence but, uniquely to non-ribosomally synthesized short peptides, several nests within them are constructed from alternating D- and L-amino acids, and such sequences seem to specially favour nests. [ABSTRACT FROM AUTHOR]

The bacterial aminopeptidase isolated from the extracellular extract of Streptomyces griseus (SGAP) is a double-zinc exopeptidase with a high preference for large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), is heat-stable, displays a high and efficient catalytic turnover and its activity is modulated by calcium ions. Several free amino acids were found to inhibit the activity of SGAP in the millimolar concentration range and can therefore serve for the study of binding of both inhibitors and reaction products. The current study is focused on the X-ray crystallographic analysis of the SGAP complexes with L-tryptophan and p-iodo-L-phenylalanine, both at 1.30 Å resolution. These two bulky inhibitory amino acids were found to bind to the active site of SGAP in very similar positions and orientations. Both of them bind to the two active-site zinc ions via their free carboxylate group, while displacing the zinc-bound water/hydroxide that is present in the native enzyme. Further stabilization of the binding of the amino-acid carboxylate group is achieved by its relatively strong interactions with the hydroxyl group of Tyr246 and the carboxylate group of Glu131. The binding is also stabilized by three specific hydrogen bonds between the amine group of the bound amino acid and enzyme residues Glu131, Asp160 and Arg202. These consistent interactions consistent the key role of these residues in the specific binding of the free amine of substrates and products, as proposed previously. The phenyl ring of Phe219 of the enzyme is involved in stacking interactions with the corresponding aromatic ring of the bound affector. This interaction seems to be important for the binding and orientation of the aromatic side chain within the specificity pocket. These structural results correlate well with the results obtained for the complexes of SGAP with other inhibitory amino acids and support the general catalytic mechanism proposed for this and related enzymes. [ABSTRACT FROM AUTHOR]

The structure of the 115 amino-acid residue protein DsvC was determined based on the anomalous scattering provided by the five S atoms present in the structure. By collecting the diffraction data at a wavelength of 1.9 Å. the anomalous signal provided by the S atoms was enhanced. However, significant radiation damage occurred during the course of the experiment, which led to differences between different parts of the data set. Only by dividing the total data set into five data sets was it possible to obtain phases; these could then he successfully extended to allow structure determination by the automated model-building program ARP/wARP. A computational correction for the radiation damage was found to significantly improve the success rate in determining the heavy-atom substructure and to improve phasing and refinement statistics. [ABSTRACT FROM AUTHOR]

KaiB is a component of the circadian clock oscillator in cyano-bacteria, which are the simplest organisms that exhibit circadian rhythms. KaiB consists of 108 amino-acid residues and has a molecular weight of 12 025 Da. KaiB and Cys-substituted KaiB mutants from the thermophilic cyanobacterium Thermosynecha coccus elongatus BP-1 were expressed as GST-fusion proteins in Escherichia coli. purified and crystallized. The crystals of wild-type KaiB belong to the monoclinic space group P21, with unit-cell parameters a = 89.6, b = 71.2, c = 106.8 Å, β = 100.1. While the native crystals diffract to 3.7 Å, osmium derivative, which show an approximately 4Å shrinkage in the b axis, diffract to 2.6 Å. the cyrstals of the singly Cys-substituted mutant T64C with Hg, which show different morphology, diffract to 2.5 Å and belong to the monoclinic space group P2, with unit-cell parameters a = 63.7, b = 33.4, c = 93.7 Å, β = 100.1. Anomalous difference Patterson maps of the Os- and Hg-derivative crystals had significant peaks in their Harker sections, suggesting that both derivatives are suitable for structure determination. [ABSTRACT FROM AUTHOR]

Focal adhesion protein 52 (FAP52) is a multidomain adaptor protein of 448 amino acids characterized as an abundant component of focal adhesions. FAP52 binds to filamin via its N-terminal α-helical domain, suggesting a role in linking focal adhesions to the actin-based cytoskeleton. The recombinant protein was crystallized using the hanging-drop vapour-diffusion method, which yielded two crystal forms. Native data were collected from both crystal forms to 2.8 and 2.1 Å resolution, respectively. For one of the crystal forms, initial MAD phasing was successfully performed using two data sets from xenon-derivatized crystals. The derivative data sets were collected using softer X-rays of 1.5 and 1.9 Å wavelength. Preliminary structural analysis reveals the presence of a dimer in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Sorcin is a 198 amino-acid Ca2+-binding protein that belongs to the penta-EF-hand family. Its Ca2+-binding domain (residues 33-198) has been crystallized in the absence of Ca2+ in two different crystal forms. Two complete data sets have been collected on a synchrotron source under cryocooling conditions from crystals grown using ammonium sulfate as precipitant: monoclinic crystals in space group C2, with unit-cell parameters a = 130.93, b = 103.85, c = 78.55 Å, β = 118.0°, diffracting to 2.1 Å, and tetragonal crystals in space group P4212, with unit-cell parameters a = b = 103.33, c = 79.15, diffracting to 2.7 Å. Crystals were also grown using PEG 6000 as precipitating agent. They also belong to space group C2, diffract to 2.8 Å and their unit-cell parameters are very similar to the first form. Structure determination by molecular replacement has been initiated. Structural information should be useful for elucidating the interaction of sorcin with membrane targets. [ABSTRACT FROM AUTHOR]

The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group I4122, with unit-cell parameters a = b = 104.5, c = 245.1 Å. Consideration of the values of VM and possible packing of the molecules within the cell suggest that the asymmetric unit contains a dimer. Examination of the behaviour of the protein on gel-filtration columns and analysis of the self-rotation function suggests that the molecule is an octamer in solution at around pH 5. Determination of the structure of this protein will provide insights into the mechanisms responsible for DNA-protein recognition at high temperature and into how the regulatory properties of the Lrp family are modified by the presence or absence of effector molecules. [ABSTRACT FROM AUTHOR]

The ToIR protein from Escherichia coli is part of the Tol-Pal multiprotein complex used by group A colicins to penetrate and kill cells. All genes of the Tol-Pal system are conserved in Gram-negative bacteria and this system is thought to play a role in the maintenance of the bacterial envelope integrity, although its exact function is not known. The TolR protein comprises 142 amino acids. The periplasmic domain of the TolR protein has been expressed, purified and crystallized. The crystals belong to the tetragonal space group P4122, with unit-cell parameters a = 46.3, c = 178.0 Å. There are one or two molecules in the asymmetric unit. Frozen crystals diffract to at least 3.2 Å resolution using synchrotron radiation. Selenomethionine substituted periplasmic TolR protein is currently being produced in order to use multiwavelength anomalous dispersion (MAD) for phasing. [ABSTRACT FROM AUTHOR]

Vinculin is a 117 kDa microfilament-associated protein located at the cytoplasmic aspects of focal contacts and cell-cell adherens type junctions. In both sites, vinculin participates in the formation of a submembrane `plaque' structure which is responsible for the attachment of actin flaments to the plasma membrane. Vinculin consists of 1066 amino acids, which form a large 90 kDa globular head domain and a rod-like 29 kDa tail domain. The two domains are separated by several stretches of proline residues where the major proteolytic cleavage sites are located. The experimental procedure for isolation and purification of vinculin from smooth muscle has been developed and crystals of native vinculin suitable for X-ray analysis have been obtained. The homogeneity of the vinculin solution was analyzed prior to crystallization using dynamic light scattering. Crystals of vinculin have been obtained in buffer containing 2 mg ml-1 protein, 0.9 M ammonium sulfate, 0.1 M MES pH 6.5 using both the hanging-drop and sitting-drop vapour-diffusion methods. The crystals have the form of rhombic plates and grow to maximal dimensions of 0.3 × 0.3 × 0.05 mm in two weeks. Preliminary X-ray data show that the crystals diffract to 3.5 Å resolution at the X11 beamline of DESY and belong to the monoclinic space group P21. Crystal unit-cell parameters are estimated to be a = 57, b = 351, c = 70 Å, α = 90, β = 113, γ = 90°. [ABSTRACT FROM AUTHOR]

The Plasmodium falciparum rab6 gene encodes a 208 amino-acid polypeptide. Two recombinant versions of P. falciparum Rab6 protein were expressed in Escherichia coli: the full-length protein and a truncated form containing residues 1-175. Both forms were purified from the soluble fraction of bacterial extract and were purified by ion-exchange chromatography and size-exclusion chromatography. Purified proteins were crystallized at pH 6.5 using the hanging-drop vapor-diffusion technique at room temperature. The full-length protein diffracted to 2.4 Å and belongs to the tetragonal space group P43212 or P41212, with unit-cell parameters a = b = 80.6, c = 90.4 Å. The crystals of the truncated protein were isomorphous with those of the full-length construct and diffracted X-rays to 2.2 Å resolution. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor (hEGF), a 6.2 kDa protein of 53 amino acids with three internal disulfide bridges, has been crystallized by the hanging-drop method. hEGF crystallizes in space group P3121 (or P3221) using MgCl2 as precipitant, with unit-cell parameters a = b = 61.4, c = 87.0 Å.Another type of crystal, obtained using NaCl as precipitant, belongs to a tetragonal point group and has unit-cell dimensions a = b = 102.5, c = 166.6 Å.The trigonal crystals with the smaller unit cell diffract X-rays better and a native data set from a single crystal has been collected to 3.0 Å resolution. [ABSTRACT FROM AUTHOR]