Orthorhombic Crystal Form (orthorhombic + crystal_form)

Distribution by Scientific Domains

Selected Abstracts

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]

Crystallization and preliminary crystallographic studies of MOMP (major outer membrane protein) from Campylobacter jejuni

Jean Michel Bolla
Campylobacter jejuni is the leading bacterial cause of human enteritis linked to ingestion of contaminated food or water. MOMP, the major outer membrane protein from these Gram-negative bacteria, belongs to the porin family. In order to determine the three-dimensional structure of this protein and to elucidate the underlying molecular mechanisms, the MOMP from C. jejuni strain 85H has been purified and crystallized by vapour diffusion. Two crystal forms were characterized for this membrane protein. X-ray diffraction data were collected to a resolution of 3.1,┼ using a synchrotron-radiation source from the orthorhombic crystal form, which belonged to space group P21212 with unit-cell parameters a = 170.1, b = 101.9, c = 104.9,┼. With a trimer in the asymmetric unit, the solvent content is 64% (VM = 3.4,┼,Da,1). The other form exhibits trigonal symmetry (space group R3) with hexagonal unit-cell parameters a = b = 94.2, c = 161.2,┼, but diffracts X-rays poorly to about 4,┼ with significant anisotropy. [source]

Purification, crystallization and molecular symmetry of CDP- d -glucose 4,6-dehydratase from Yersinia pseudotuberculosis

Erik M. Vogan
The enzyme CDP- d -glucose 4,6-dehydratase (EC is an NAD+ -dependent oxidoreductase which catalyzes the irreversible conversion of CDP- d -glucose to CDP-4-keto-6-deoxy- d -glucose. The product of this reaction is an intermediate in the synthesis of all CDP-şlinked 3,6-dideoxyhexoses, an important class of antigenic determinants found in the lipopolysaccharide layer of Gram-negative bacteria. Crystals of a recombinant form of this enzyme from Yersinia pseudotuberculosis have been grown in two crystal forms, both possessing pseudo-translational non-crystallographic symmetry, with dramatically different diffraction characteristics. A complete 1.8,┼ data set has been collected from the primitive orthorhombic crystal form, for which the non-crystallographic symmetry is described in detail. [source]

Crystallization of agGST1-6, a recombinant glutathione S -transferase from a DDT-resistant strain of Anopheles gambiae

Pamela H. Roberts
Glutathione S -transferases (GSTs) belong to a family of detoxification enzymes that conjugate glutathione to various xenobiotics, thus facilitating their expulsion from the cell. GST activity is elevated in many insecticide-resistant insects, including the DDT-resistant malaria vector Anopheles gambiae. Crystals of the recombinant form of a GST from A. gambiae, agGST1-6, have been grown in at least five different crystal forms, with a broad range of diffraction resolution limits. A complete 2.0,┼ data set has been collected on a C -centered orthorhombic crystal form with unit-cell parameters a = 99.0, b = 199.4, c = 89.6,┼. A search for heavy-atom derivatives has been initiated, along with phase-determination efforts by molecular replacement. [source]

Structures of feline immunodeficiency virus dUTP pyrophosphatase and its nucleotide complexes in three crystal forms

G. Sridhar Prasad
dUTP pyrophosphatase (dUTPase) cleaves the ,-, phosphodiester of dUTP to form pyrophosphate and dUMP, preventing incorporation of uracil into DNA and providing the substrate for thymine synthesis. Seven crystal structures of feline immunodeficiency virus (FIV) dUTPase in three crystal forms have been determined, including complexes with substrate (dUTP), product (dUMP) or inhibitor (dUDP) bound. The native enzyme has been refined at 1.40,┼ resolution in a hexagonal crystal form and at 2.3,┼ resolution in an orthorhombic crystal form. In the dUDP complex in a cubic crystal form refined at 2.5,┼ resolution, the C-terminal conserved P-loop motif is fully ordered. The analysis defines the roles of five sequence motifs in interaction with uracil, deoxyribose and the ,-, ,- and ,-phosphates. The enzyme utilizes adaptive recognition to bind the ,- and ,-phosphates. In particular, the ,-, phosphodiester adopts an unfavorable eclipsed conformation in the presence of the P-loop. This conformation may be relevant to the mechanism of ,-, phosphodiester bond cleavage. [source]

The 1.30,┼ resolution structure of the Bacillus subtilis chorismate mutase catalytic homotrimer

Jane E. Ladner
The crystal structure of the Bacillus subtilis chorismate mutase, an enzyme of the aromatic amino acids biosynthetic pathway, was determined to 1.30,┼ resolution. The structure of the homotrimer was determined by molecular replacement using orthorhombic crystals of space group P212121 with unit-cell parameters a = 52.2, b = 83.8, c = 86.0,┼. The ABC trimer of the monoclinic crystal structure [Chook et al. (1994), J. Mol. Biol.240, 476,500] was used as the starting model. The final coordinates are composed of three complete polypeptide chains of 127 amino-acid residues. In addition, there are nine sulfate ions, five glycerol molecules and 424 water molecules clearly visible in the structure. This structure was refined with aniosotropic temperature factors, has excellent geometry and a crystallographic R factor of 0.169 with an Rfree of 0.236. The three active sites of the macromolecule are at the subunit interfaces, with residues from two subunits contributing to each site. This orthorhombic crystal form was grown using ammonium sulfate as the precipitant; glycerol was used as a cryoprotectant during data collection. A glycerol molecule and sulfate ion in each of the active sites was found mimicking a transition-state analog. In this structure, the C-terminal tails of the subunits of the trimer are hydrogen bonded to residues of the active site of neighboring trimers in the crystal and thus cross-link the molecules in the crystal lattice. [source]

Preliminary crystallographic analysis of the N-terminal domain of FILIA, a protein essential for embryogenesis

Juke Wang
FILIA is a component of the subcortical maternal complex that is essential for early stage embryogenesis. Its 6ÎHis-tagged N-terminal domain was expressed in Escherichia coli and purified to homogeneity. Two types of crystals formed under different crystallization conditions during screening. Orthorhombic crystals appeared in a solution containing 1.4,M ammonium sulfate, 0.1,M Tris pH 8.2 and 12% glycerol, while tetragonal crystals were obtained using 15% PEG 4000 mixed with 0.1,M HEPES pH 7.5 and 15% 2-propanol. High-quality diffraction data were collected from the two crystal forms to resolutions of 1.8 and 2.2,┼, respectively, using synchrotron radiation. The Matthews coefficients indicated that the P212121 and P41212 crystals contained two molecules and one molecule per asymmetric unit, respectively. A selenomethionine-substituted sample failed to crystallize under the native conditions, but another orthorhombic crystal form was obtained under different conditions and anomalous diffraction data were collected. [source]

Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Mrs2, a magnesium ion transporter from yeast inner mitochondrial membrane

Muhammad Bashir Khan
Mrs2 transporters are distantly related to the major bacterial Mg2+ transporter CorA and to Alr1, which is found in the plasma membranes of lower eukaryotes. Common features of all Mrs2 proteins are the presence of an N-terminal soluble domain followed by two adjacent transmembrane helices (TM1 and TM2) near the C-terminus and of the highly conserved F/Y-G-M-N sequence motif at the end of TM1. The inner mitochondrial domain of the Mrs2 from Saccharomyces cerevisae was overexpressed, purified and crystallized in two different crystal forms corresponding to an orthorhombic and a hexagonal space group. The crystals diffracted X-rays to 1.83 and 4.16,┼ resolution, respectively. Matthews volume calculations suggested the presence of one molecule per asymmetric unit in the orthorhombic crystal form and of five or six molecules per asymmetric unit in the hexagonal crystal form. The phase problem was solved for the orthorhombic form by a single-wavelength anomalous dispersion experiment exploiting the sulfur anomalous signal. [source]

Crystallization and preliminary X-ray analysis of the human respiratory syncytial virus nucleocapsid protein

K. El Omari
Human respiratory syncytial virus (HRSV) has a nonsegmented negative-stranded RNA genome which is encapsidated by the HRSV nucleocapsid protein (HRSVN) that is essential for viral replication. HRSV is a common cause of respiratory infection in infants, yet no effective antiviral drugs to combat it are available. Recent data from an experimental anti-HRSV compound, RSV-604, indicate that HRSVN could be the target site for drug action. Here, the expression, purification and preliminary data collection of decameric HRSVN as well as monomeric N-terminally truncated HRSVN mutants are reported. Two different crystal forms of full-length selenomethionine-labelled HRSVN were obtained that diffracted to 3.6 and ,5,┼ resolution and belonged to space group P212121, with unit-cell parameters a = 133.6, b = 149.9, c = 255.1,┼, and space group P21, with unit-cell parameters a = 175.1, b = 162.6, c = 242.8,┼, , = 90.1░, respectively. For unlabelled HRSVN, only crystals belonging to space group P21 were obtained that diffracted to 3.6,┼. A self-rotation function using data from the orthorhombic crystal form confirmed the presence of tenfold noncrystallographic symmetry, which is in agreement with a reported electron-microscopic reconstruction of HRSVN. Monomeric HRSVN generated by N-terminal truncation was designed to assist in structure determination by reducing the size of the asymmetric unit. Whilst such HRSVN mutants were monomeric in solution and crystallized in a different space group, the size of the asymmetric unit was not reduced. [source]

Structure of the catalytic trimer of Methanococcus jannaschii aspartate transcarbamoylase in an orthorhombic crystal form

Jacqueline Vitali
Crystals of the catalytic subunit of Methanococcus jannaschii aspartate transcarbamoylase in an orthorhombic crystal form contain four crystallographically independent trimers which associate in pairs to form stable staggered complexes that are similar to each other and to a previously determined monoclinic C2 form. Each subunit has a sulfate in the central channel. The catalytic subunits in these complexes show flexibility, with the elbow angles of the monomers differing by up to 7.4░ between crystal forms. Moreover, there is also flexibility in the relative orientation of the trimers around their threefold axis in the complexes, with a difference of 4░ between crystal forms. [source]

Structure of 3(17),-hydroxysteroid dehydrogenase (AKR1C21) holoenzyme from an orthorhombic crystal form: an insight into the bifunctionality of the enzyme

Urmi Dhagat
Mouse 3(17),-hydroxysteroid dehydrogenase (AKR1C21) is a bifunctional enzyme that catalyses the oxidoreduction of the 3- and 17-hydroxy/keto groups of steroid substrates such as oestrogens, androgens and neurosteroids. The structure of the AKR1C21,NADPH binary complex was determined from an orthorhombic crystal belonging to space group P212121 at a resolution of 1.8,┼. In order to identify the factors responsible for the bifunctionality of AKR1C21, three steroid substrates including a 17-keto steroid, a 3-keto steroid and a 3,-hydroxysteroid were docked into the substrate-binding cavity. Models of the enzyme,coenzyme,substrate complexes suggest that Lys31, Gly225 and Gly226 are important for ligand recognition and orientation in the active site. [source]

Monoclinic crystal form of Aspergillus niger,-şamylase in complex with maltose at 1.8,┼ resolution

A. Vuji
Aspergillus niger,-amylase catalyses the hydrolysis of ,-1,4-glucosidic bonds in starch. It shows 100% sequence identity to the A. oryzae homologue (also called TAKA-amylase), three crystal structures of which have been published to date. Two of them belong to the orthorhombic space group P212121 with one molecule per asymmetric unit and one belongs to the monoclinic space group P21 with three molecules per asymmetric unit. Here, the purification, crystallization and structure determination of A. niger,-amylase crystallized in the monoclinic space group P21 with two molecules per asymmetric unit in complex with maltose at 1.8,┼ resolution is reported. Furthermore, a novel 1.6,┼ resolution orthorhombic crystal form (space group P21212) of the native enzyme is presented. Four maltose molecules are observed in the maltose,,-amylase complex. Three of these occupy active-site subsites ,2 and ,1, +1 and +2 and the hitherto unobserved subsites +4 (Asp233, Gly234) and +5 (Asp235). The fourth maltose molecule binds at the distant binding sites d1 (Tyr382) and d2 (Trp385), also previously unobserved. Furthermore, it is shown that the active-site groove permits different binding modes of sugar units at subsites +1 and +2. This flexibility of the active-site cleft close to the catalytic centre might be needed for a productive binding of substrate chains and/or release of products. [source]

Crystallization and initial X-ray diffraction studies of scaffolding protein (gp7) of bacteriophage ,29

Dwight L. Anderson
The Bacillus subtilis bacteriophage ,29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293,K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P41212, with unit-cell parameters a = b = 77.13, c = 37.12,┼. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34,┼. Complete data sets have been collected to 1.78 and 1.80,┼ for forms I and II, respectively, at 100,K using Cu,K, X-rays from a rotating-anode generator. Calculation of a VM value of 2.46,┼3,Da,1 for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a VM of 4.80,┼3,Da,1 with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7. [source]

The structure of dihydrodipicolinate reductase (DapB) from Mycobacterium tuberculosis in three crystal forms

Robert Janowski
Dihydrodipicolinate reductase (DHDPR, DapB) is an enzyme that belongs to the l -lysine biosynthetic pathway. DHDPR reduces the ,,,-unsaturated cyclic imine 2,3-dihydrodipicolinic acid to yield the compound 2,3,4,5-tetrahydrodipicolinic acid in a pyridine nucleotide-dependent reaction. The substrate of this reaction is the unstable product of the preceding enzyme dihydrodipicolinate synthase (DHDPS, DapA). Here, the structure of apo-DHDPR from Mycobacterium tuberculosis is reported in two orthorhombic crystal forms, as well as the structure of DHDPR from M. tuberculosis in complex with NADH in a monoclinic crystal form. A comparison of the results with previously solved structures of this enzyme shows that DHDPR undergoes a major conformational change upon binding of its cofactor. This conformational change can be interpreted as one of the low-frequency normal modes of the structure. [source]

Purification, crystallization and preliminary crystallographic study of low oxygen-affinity haemoglobin from cat (Felis silvestris catus) in two different crystal forms

M. Balasubramanian
Haemoglobin is a metalloprotein which plays a major role in the transportation of oxygen from the lungs to tissues and of carbon dioxide back to the lungs. The present work reports the preliminary crystallographic study of low oxygen-affinity haemoglobin from cat in different crystal forms. Cat blood was collected, purified by anion-exchange chromatography and crystallized in two different conditions by the hanging-drop vapour-diffusion method under unbuffered low-salt and buffered high-salt concentrations using PEG 3350 as a precipitant. Intensity data were collected using MAR345 and MAR345dtb image-plate detector systems. Cat haemoglobin crystallizes in monoclinic and orthorhombic crystal forms with one and two whole biological molecules (,2,2), respectively, in the asymmetric unit. [source]

Crystallization and preliminary crystallographic analysis of cgHle, a homoserine acetyltransferase homologue, from Corynebacterium glutamicum

Christine T÷lzer
CgHle is an enzyme that is encoded by gene cg0961 from Corynebacterium glutamicum. The physiological function of cgHle is so far unclear. Bioinformatic annotations based on sequence homology indicated that cgHle may be an acetyl-CoA:homoserine acetyl transferase and as such may be involved in methionine biosynthesis, but recent evidence has shown that it is an esterase that catalyzes the hydrolysis of acetyl esters. Here, the crystallization of cgHle in two orthorhombic crystal forms, a trigonal crystal form and a monoclinic crystal form is described. The trigonal crystals have a solvent content of 83.7%, which is one of the highest solvent contents ever found for protein crystals. One of the orthorhombic crystals diffracted X-rays to at least 1.2,┼ resolution. [source]