New Crystal Form (new + crystal_form)

Distribution by Scientific Domains

Selected Abstracts

A new polymorph of 2-methyl-6-nitroaniline

Samantha K. Callear
A new crystal form of 2-methyl-6-nitroaniline, C7H8N2O2, crystallizing with Z, = 2 in the space group P21/c, has been identified during screening for salts and cocrystals. The different N,H...O hydrogen-bonding synthons result in linear V-shaped chains in the new polymorph, rather than the helical chain arrangement seen in the known form where Z, = 1. The presence of a second component during crystallization appears to have determined the resultant crystal form of 2-methyl-6-nitroaniline. [source]

Structure analysis of endosialidase NF at 0.98,┼ resolution

Eike C. Schulz
Endosialidase NF (endoNF) is a bacteriophage-derived endosialidase that specifically degrades ,-2,8-linked polysialic acid. The structure of a new crystal form of endoNF in complex with sialic acid has been refined at 0.98,┼ resolution. The 210,kDa homotrimeric multi-domain enzyme displays outstanding stability and resistance to SDS. Even at atomic resolution, only a minor fraction of side chains possess alternative conformations. However, multiple conformations of an active-site residue imply that it has an important catalytic function in the cleavage mechanism of polysialic acid. [source]

A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavity

Daniel A. Breustedt
Tear lipocalin (TLC) with the bound artificial ligand 1,4-butanediol has been crystallized in space group P21 with four protein molecules in the asymmetric unit and its X-ray structure has been solved at 2.6,┼ resolution. TLC is a member of the lipocalin family that binds ligands with diverse chemical structures, such as fatty acids, phospholipids and cholesterol as well as microbial siderophores and the antibiotic rifampin. Previous X-ray structural analysis of apo TLC crystallized in space group C2 revealed a rather large bifurcated ligand pocket and a partially disordered loop region at the entrace to the cavity. Analysis of the P21 crystal form uncovered major conformational changes (i) in ,-strands B, C and D, (ii) in loops 1, 2 and 4 at the open end of the ,-barrel and (iii) in the extended C-terminal segment, which is attached to the ,-barrel via a disulfide bridge. The structural comparison indicates high conformational plasticity of the loop region as well as of deeper parts of the ligand pocket, thus allowing adaptation to ligands that differ vastly in size and shape. This illustrates a mechanism for promiscuity in ligand recognition which may also be relevant for some other physiologically important members of the lipocalin protein family. [source]

Structure of isochorismate synthase in complex with magnesium

James F. Parsons
The electron carrier menaquinone is one of many important bacterial metabolites that are derived from the key intermediate chorismic acid. MenF, the first enzyme in the menaquinone pathway, catalyzes the isomerization of chorismate to isochorismate. Here, an improved structure of MenF in a new crystal form is presented. The structure, solved at 2.0,┼ resolution in complex with magnesium, reveals a well defined closed active site. Existing evidence suggests that the mechanism of the reaction catalyzed by MenF involves nucleophilic attack of a water molecule on the chorismate ring. The structure reveals a well defined water molecule located in an appropriate position for activation by Lys190 and attack on the substrate. [source]

Surface-entropy reduction approaches to manipulate crystal forms of ,-ketoacyl acyl carrier protein synthase II from Streptococcus pneumoniae

Gopalakrishnan Parthasarathy
A series of experiments with ,-ketoacyl acyl carrier protein synthase II (FabF) from Streptococcus pneumonia (spFabF) were undertaken to evaluate the capability of surface-entropy reduction (SER) to manipulate protein crystallization. Previous work has shown that this protein crystallizes in two forms. The triclinic form contains four molecules in the asymmetric unit (a.u.) and diffracts to 2.1,┼ resolution, while the more desirable primitive orthorhombic form contains one molecule in the a.u. and diffracts to 1.3,┼. The aim was to evaluate the effect of SER mutations that were specifically engineered to avoid perturbing the crystal-packing interfaces employed by the favorable primitive orthorhombic crystal form while potentially disrupting a surface of the protein employed by the less desirable triclinic crystal form. Two mutant proteins were engineered, each of which harbored five SER mutations. Extensive crystallization screening produced crystals of the two mutants, but only under conditions that differed from those used for the native protein. One of the mutant proteins yielded crystals that were of a new form (centered orthorhombic), despite the fact that the interfaces employed by the primitive orthorhombic form of the native protein were specifically unaltered. Structure determination at 1.75,┼ resolution reveals that one of the mutations, E383A, appears to play a key role in disfavouring the less desirable triclinic crystal form and in generating a new surface for a packing interaction that stabilizes the new crystal form. [source]

High-resolution structure of human cytoglobin: identification of extra N- and C-termini and a new dimerization mode

Masatomo Makino
Cytoglobin (Cgb) is a recently discovered member of the vertebrate haem-containing globin family. The structure of a new crystal form of wild-type human Cgb (space group C2) was determined at a resolution of 1.68,┼. The results show the presence of an additional helix in the N-terminal residues (4,ş20) prior to the A helix and an ordered loop structure in the C-terminal region (168,188), while these extended peptides were invisible owing to disorder in the previously reported structures using a P3221 crystal at a resolution of 2.4,┼. A detailed comparison of the two crystal structures shows differences in the conformation of the residues (i.e. Arg84) in the haem environment owing to a different dimeric arrangement. [source]

A new crystal form of XT6 enables a significant improvement of its diffraction quality and resolution

Maya Bar
Xylanases (1,4-,- d -xylan xylanhydrolases; EC hydrolyze the 1,4-,- d -xylopyranosyl linkage of xylans. The detailed structural characterization of these enzymes is of interest for the elucidation of their catalytic mechanism and for their rational modification toward improved stability and specificity. An extracellular xylanase from Geobacillus stearothermophilus T-6 (XT6) has recently been cloned, overexpressed, purified and biochemically characterized. Previous crystallographic efforts resulted in a hexagonal crystal form, which subsequently proved to be of limited use for structural analysis, mainly because of its relatively poor diffraction quality and resolution. A systematic search for more suitable crystals of XT6 recently resulted in a new crystal form of this enzyme with significantly improved diffraction characteristics. The new crystals belong to a C -centred monoclinic crystal system (space group C2), with unit-cell parameters a = 121.5, b = 61.7, c = 89.1,┼, , = 119.7░. These crystals diffract X-rays to better than 1.5,┼ resolution, showing a very clear diffraction pattern of relatively high quality. The crystals are mechanically strong and exhibit excellent radiation-stability when frozen under cold nitrogen gas. A full diffraction data set to 1.45,┼ resolution (94.1% completeness, Rmerge = 7.0%) has been collected from flash-frozen crystals of the native enzyme at 95,K using synchrotron radiation. Crystals of the E159A/E265A catalytic double mutant of XT6 were found to be isomorphous to those of native XT6. They were used for a full measurement of 1.8,┼ resolution diffraction data at 100,K (90.9% completeness; Rmerge = 5.0%). These data are currently being used for the high-resolution structure determination of XT6 and its mutant for mechanistic interpretations and rational introduction of thermostability. [source]

Structural comparison of Escherichia colil -asparaginase in two monoclinic space groups

Mario Sanches
The functional l -asparaginase from Escherichia coli is a homotetramer with a molecular weight of about 142,kDa. The X-ray structure of the enzyme, crystallized in a new form (space group C2) and refined to 1.95,┼ resolution, is compared with that of the previously determined crystal form (space group P21). The asymmetric unit of the new crystal form contains an l -asparaginase dimer instead of the tetramer found in the previous crystal form. It is found that crystal contacts practically do not affect the conformation of the protein. It is shown that subunit C of the tetrameric form is in a conformation which is systematically different from that of all other subunits in both crystal forms. Major conformational differences are confined to the lid loop (residues 14,27). In addition, the stability of this globular protein is analyzed in terms of the interactions between hydrophobic parts of the subunits. [source]

Structure of a new crystal form of tetraubiquitin

Cynthia L. Phillips
Polyubiquitin chains, in which the C-terminus and a lysine side chain of successive ubiquitin molecules are linked by an isopeptide bond, function to target substrate proteins for degradation by the 26S proteasome. Chains of at least four ubiquitin moieties appear to be required for efficient recognition by the 26S proteasome, although the conformations of the polyubiquitin chains recognized by the proteasome or by other enzymes involved in ubiquitin metabolism are currently unknown. A new crystal form of tetraubiquitin, which has two possible chain connectivities that are indistinguishable in the crystal, is reported. In one possible connectivity, the tetraubiquitin chain is extended and packs closely against the antiparallel neighbor chain in the crystal to conceal a hydrophobic surface implicated in 26S proteasome recognition. In the second possibility, the tetraubiqutitin forms a closed compact structure, in which that same hydrophobic surface is buried. Both of these conformations are quite unlike the structure of tetraubiquitin that was previously determined in a different crystal form [Cook et al. (1994), J. Mol. Biol.236, 601,609]. The new structure suggests that polyubiquitin chains may possess a substantially greater degree of conformational flexibility than has previously been appreciated. [source]

Crystallization and preliminary crystallographic studies of a new crystal form of Escherichia colil -şasparaginase II (Ser58Ala mutant)

Maciej Kozak
Periplasmic Escherichia colil -asparaginase II with an Ser58Ala mutation in the active-site cavity has been crystallized in a new orthorhombic form (space group P21212). Crystals of this polymorph suitable for X-ray diffraction have been obtained by vapour diffusion using two sets of conditions: (i) 1% agarose gel using MPD as precipitant (pH 4.8) and (ii) liquid droplets using PEG-MME 550 (pH 9.0). The crystals grown in agarose gel are characterized by unit-cell parameters a = 226.9, b = 128.4, c = 61.9,┼ and diffract to 2.3,┼ resolution. The asymmetric unit contains six protein molecules arranged into one pseudo-222-symmetric homotetramer and an active-site competent dimer from which another homotetramer is generated by crystallographic symmetry. [source]

Near-atomic resolution analysis of BipD, a component of the type III secretion system of Burkholderia pseudomallei

M. Pal
Burkholderia pseudomallei, the causative agent of melioidosis, possesses a type III protein secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to inject virulence-associated proteins into target cells of the host organism. The bipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and is most likely to be functionally analogous to IpaD from Shigella and SipD from Salmonella. Proteins in this family are thought to act as extracellular chaperones at the tip of the secretion needle to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and may also link the translocon pore with the secretion needle. BipD has been crystallized in a monoclinic crystal form that diffracted X-rays to 1.5,┼ resolution and the structure was refined to an R factor of 16.1% and an Rfree of 19.8% at this resolution. The putative dimer interface that was observed in previous crystal structures was retained and a larger surface area was buried in the new crystal form. [source]

A new crystal form of Lys48-linked diubiquitin

Jean-Franšois Trempe
Lys48-linked polyubiquitin chains are recognized by the proteasome as a tag for the degradation of the attached substrates. Here, a new crystal form of Lys48-linked diubiquitin (Ub2) was obtained and the crystal structure was refined to 1.6,┼ resolution. The structure reveals an ordered isopeptide bond in a trans configuration. All three molecules in the asymmetric unit were in the same closed conformation, in which the hydrophobic patches of both the distal and the proximal moieties interact with each other. Despite the different crystallization conditions and different crystal packing, the new crystal structure of Ub2 is similar to the previously published structure of diubiquitin, but differences are observed in the conformation of the flexible isopeptide linkage. [source]

A new crystal form of human diamine oxidase

Aaron P. McGrath
Copper amine oxidases (CAOs) are ubiquitous in nature and catalyse the oxidative deamination of primary amines to the corresponding aldehydes. Humans have three viable CAO genes (AOC1,3). AOC1 encodes human diamine oxidase (hDAO), which is the frontline enzyme for histamine metabolism. hDAO is unique among CAOs in that it has a distinct substrate preference for diamines. The structure of hDAO in space group P212121 with two molecules in the asymmetric unit has recently been reported. Here, the structure of hDAO refined to 2.1,┼ resolution in space group C2221 with one molecule in the asymmetric unit is reported. [source]

Crystallization and preliminary X-ray crystallographic analysis of a new crystal form of hydroxylamine oxidoreductase from Nitrosomonas europaea

Peder E. Cedervall
Hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea is a homotrimeric protein that catalyzes the oxidation of hydroxylamine to nitrite. Each monomer, with a molecular weight of 67.1,kDa, contains seven c -type hemes and one heme P460, the porphyrin ring of which is covalently linked to a tyrosine residue from an adjacent subunit. HAO was first crystallized and structurally characterized at a resolution of 2.8,┼ in 1997. The structure was solved in space group P63 and suffered from merohedral twinning. Here, a crystallization procedure is presented that yielded untwinned crystals belonging to space group P21212, which diffracted to 2.25,┼ resolution and contained one trimer in the asymmetric unit. The unit-cell parameters were a = 140.7, b = 142.6, c = 107.4,┼. [source]

Structure of an Escherichia coli N -acetyl- d -neuraminic acid lyase mutant, E192N, in complex with pyruvate at 1.45,┼ resolution

Ivan Campeotto
The structure of a mutant variant of Escherichia coli N -acetyl- d -neuraminic acid lyase (NAL), E192N, in complex with pyruvate has been determined in a new crystal form. It crystallized in space group P212121, with unit-cell parameters a = 78.3, b = 108.5, c = 148.3,┼. Pyruvate has been trapped in the active site as a Schiff base with the catalytic lysine (Lys165) without the need for reduction. Unlike the previously published crystallization conditions for the wild-type enzyme, in which a mother-liquor-derived sulfate ion is strongly bound in the catalytic pocket, the low-salt conditions described here will facilitate the determination of further E. coli NAL structures in complex with other active-site ligands. [source]

Crystallization and preliminary X-ray analysis of the small subunit (R2F) of native ribonucleotide reductase from Corynebacterium ammoniagenes

Hideaki Ogata
Ribonucleotide reduction, the unique step in DNA-precursor biosynthesis, involves radical-dependent redox chemistry and diverse metallo-cofactors. The metallo-cofactor (R2F) encoded by the nrdF (nucleotide reduction) gene in Corynebacterium ammoniagenes ATCC 6872 was isolated after homologous expression and a new crystal form of ribonucleotide reductase R2F was obtained. R2F was crystallized at 277,K using the vapour-diffusion method with PEG as the precipitating agent. A data set was collected to 1.36,┼ resolution from a single crystal at 100,K using synchrotron radiation. The crystal belonged to space group C2, with unit-cell parameters a = 96.21, b = 87.68, c = 83.25,┼, , = 99.29░. The crystal contained two molecules per asymmetric unit, with a Matthews coefficient (VM) of 2.69,┼3,Da,1; the solvent content was estimated to be 54.3%. X-ray fluorescence spectroscopy and MAD diffraction data indicated the presence of manganese in the molecule and the absence of iron. [source]

A new crystal form of bovine pancreatic RNase A in complex with 2,-deoxyguanosine-5,-monophosphate

Steven B. Larson
The structure of bovine pancreatic RNase A has been determined in complex with 2,-deoxyguanosine-5,-monophosphate (dGMP) at 1.33,┼ resolution at room temperature in a previously unreported unit cell belonging to space group P31. There are two molecules of nucleotide per enzyme molecule, one of which lies in the active-site cleft in the productive binding mode, whilst the guanine base of the other dGMP occupies the pyrimidine-specific binding site in a nonproductive mode such that it forms hydrogen bonds to the phosphate group of the first dGMP. This is the first RNase A structure containing a guanine base in the B2 binding site. Each dGMP molecule is involved in intermolecular interactions with adjacent RNase A molecules in the lattice and the two nucleotides appear to direct the formation of the crystal lattice. Because GMP may be produced during degradation of RNA, this association could represent an inhibitor complex and thereby affect the observed enzyme kinetics. [source]

The copper-containing amine oxidase from Arthrobacter globiformis: refinement at 1.55 and 2.20,┼ resolution in two crystal forms

David B. Langley
Copper-containing amine oxidases are found in all the major kingdoms of life. They catalyse the oxidation of organic amines in the presence of molecular dioxygen to aldehydes and hydrogen peroxide. The catalytic centres contain a Cu atom and a topaquinone cofactor formed autocatalytically from a tyrosine residue in the presence of Cu and molecular oxygen. The structure of the Cu-containing amine oxidase from Arthrobacter globiformis, which was previously refined at 1.8,┼ resolution in space group C2 with unit-cell parameters a = 157.84, b = 63.24, c = 91.98,┼, , = 112.0░ [Wilce et al. (1997), Biochemistry, 36, 16116,16133], has been re-refined with newly recorded data at 1.55,┼ resolution. The structure has also been solved and refined at 2.2,┼ resolution in a new crystal form, space group C2, with unit-cell parameters a = 158.04, b = 64.06, c = 69.69,┼, , = 111.7░. [source]

Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae

Emerich Mihai Gazdag
The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30,mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5,mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research. [source]

Improvement of the quality of lumazine synthase crystals by protein engineering

Lidia RodrÝguez-Fernßndez
Icosahedral macromolecules have a wide spectrum of potential nanotechnological applications, the success of which relies on the level of accuracy at which the molecular structure is known. Lumazine synthase from Bacillus subtilis forms a 150,┼ icosahedral capsid consisting of 60 subunits and crystallizes in space group P6322 or C2. However, the quality of these crystals is poor and structural information is only available at 2.4,┼ resolution. As classical strategies for growing better diffracting crystals have so far failed, protein engineering has been employed in order to improve the overexpression and purification of the molecule as well as to obtain new crystal forms. Two cysteines were replaced to bypass misfolding problems and a charged surface residue was replaced to force different molecular packings. The mutant protein crystallizes in space group R3, with unit-cell parameters a = b = 313.02, c = 365.77,┼, , = , = 90.0, , = 120░, and diffracts to 1.6,┼ resolution. [source]