scholarly journals Crystallization and preliminary crystallographic analysis of LipC12, a true lipase isolated through a metagenomics approach

2012 ◽  
Vol 68 (2) ◽  
pp. 175-177 ◽  
Author(s):  
V. P. Martini ◽  
A. Glogauer ◽  
J. Iulek ◽  
E. M. Souza ◽  
F. O. Pedrosa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Three Dimensional ◽  
Sodium Formate ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Sequence Identity

LipC12, a true lipase from family I.1 of bacterial lipases which was previously isolated through a metagenomics approach, contains 293 amino acids. Among lipases of known three-dimensional structure, it has a sequence identity of 47% to the lipase fromPseudomonas aeruginosaPAO1. Recombinant N-terminally His6-tagged LipC12 protein was expressed inEscherichia coli, purified in a homogenous form and crystallized in several conditions, with the best crystals being obtained using 2.0 Msodium formate and 0.1 Mbis-tris propane pH 7.0. X-ray diffraction data were collected to 2.70 Å resolution. The crystals belonged to the tetragonal space groupP4122, with unit-cell parametersa=b= 58.62,c = 192.60 Å.

scholarly journals Crystallization and preliminary crystallographic studies of PotA, a membrane-associated ATPase of the spermidine-preferential uptake system inThermotoga maritima

2014 ◽  
Vol 70 (6) ◽  
pp. 738-741 ◽  
Author(s):  
Shigeru Sugiyama ◽  
Keiko Kashiwagi ◽  
Keisuke Kakinouchi ◽  
Hideyuki Tomitori ◽  
Ken Kanai ◽  
...  
Keyword(s):  
Thermotoga Maritima ◽  
Three Dimensional ◽  
Crystallographic Analysis ◽  
Uptake System ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Polyamine Uptake ◽  
Preferential Uptake

A membrane-associated ATPase, PotA, is a component of the spermidine-preferential uptake system in prokaryotes that plays an important role in normal cell growth by regulating the cellular polyamine concentration. No three-dimensional structures of membrane-associated ATPases in polyamine-uptake systems have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of PotA fromThermotoga maritimaare reported. Diffraction data were collected and processed to 2.7 Å resolution from both native and selenomethionine-labelled crystals. Preliminary crystallographic analysis revealed that the crystals belonged to the hexagonal space groupP3112 (orP3212), with unit-cell parametersa=b= 88.9,c= 221.2 Å, α = 90, β = 90, γ = 120°, indicating that a dimer was present in the asymmetric unit.

scholarly journals Cloning, purification, crystallization and preliminary X-ray diffraction crystallographic study of acyl-protein thioesterase 1 fromSaccharomyces cerevisiae

2012 ◽  
Vol 68 (7) ◽  
pp. 775-777
Author(s):  
Ye Yuan ◽  
Xiao Wang ◽  
Xu Li ◽  
Maikun Teng ◽  
Liwen Niu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Modification ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Crystallographic Study ◽  
Preliminary Model

Palmitoylation/depalmitoylation plays an important role in protein modification. yApt1 is the only enzyme inSaccharomyces cerevisiaethat catalyses depalmitoylation. In the present study, recombinant full-length yApt1 was cloned, expressed, purified and crystallized. The crystals diffracted to 2.40 Å resolution and belonged to space groupP42212, with unit-cell parametersa = b = 146.43,c = 93.29 Å. A preliminary model of the three-dimensional structure has been built and further refinement is ongoing.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of an artificial molten-globular-like triosephosphate isomerase protein of mixed phylogenetic origin

2014 ◽  
Vol 70 (11) ◽  
pp. 1521-1525 ◽  
Author(s):  
Venuka Durani Goyal ◽  
Pooja Yadav ◽  
Ashwani Kumar ◽  
Biplab Ghosh ◽  
Ravindra D. Makde
Keyword(s):  
Triosephosphate Isomerase ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Sequence Identity ◽  
Phylogenetic Origin ◽  
Novel Protein

A bioinformatics-based protein-engineering approach called consensus design led to the construction of a chimeric triosephosphate isomerase (TIM) protein called ccTIM (curated consensus TIM) which is as active asSaccharomyces cerevisiaeTIM despite sharing only 58% sequence identity with it. The amino-acid sequence of this novel protein is as identical to native sequences from eukaryotes as to those from prokaryotes and shares some biophysical traits with a molten globular protein. Solving its crystal structure would help in understanding the physical implications of its bioinformatics-based sequence. In this report, the ccTIM protein was successfully crystallized using the microbatch-under-oil method and a full X-ray diffraction data set was collected to 2.2 Å resolution using a synchrotron-radiation source. The crystals belonged to space groupC2221, with unit-cell parametersa= 107.97,b= 187.21,c= 288.22 Å. Matthews coefficient calculations indicated the presence of six dimers in the asymmetric unit, with an approximate solvent content of 46.2%.

scholarly journals Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Zucchini fromDrosophila melanogaster

2012 ◽  
Vol 68 (11) ◽  
pp. 1346-1350
Author(s):  
Satoshi Fukuhara ◽  
Hiroshi Nishimasu ◽  
Luc Bonnefond ◽  
Naoki Matsumoto ◽  
Ryuichiro Ishitani ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phospholipase D ◽  
Germ Cells ◽  
Underlying Mechanism ◽  
Crystallographic Analysis ◽  
Genomic Integrity ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters

PIWI-interacting RNAs (piRNAs) bind PIWI proteins and silence transposons to maintain the genomic integrity of germ cells. Zucchini (Zuc), a phospholipase D superfamily member, is conserved among animals and is implicated in piRNA biogenesis. However, the underlying mechanism by which Zuc participates in piRNA biogenesis remains elusive.Drosophila melanogasterZuc (DmZuc) was expressed inEscherichia coli, purified and crystallized. X-ray diffraction data were collected to 1.75 Å resolution. The crystal belonged to space groupP21, with unit-cell parametersa= 55.0,b= 71.2,c= 56.3 Å, β = 107.9°.

scholarly journals Purification, crystallization and preliminary crystallographic analysis of a 4-thiouridine synthetase–RNA complex

2013 ◽  
Vol 69 (4) ◽  
pp. 421-424 ◽  
Author(s):  
Peter-Thomas Naumann ◽  
Charles T. Lauhon ◽  
Ralf Ficner
Keyword(s):  
Diffraction Data ◽  
Thermotoga Maritima ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Dependent Modification ◽  
Rna Complex

The sulfurtransferase 4-thiouridine synthetase (ThiI) is involved in the ATP-dependent modification of U8 in tRNA. ThiI fromThermotoga maritimawas cloned, overexpressed and purified. A complex comprising ThiI and a truncated tRNA was prepared and crystallized, and X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 102.9,b= 112.8,c= 132.8 Å.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of human myotubularin-related protein 1

2015 ◽  
Vol 71 (3) ◽  
pp. 261-265 ◽  
Author(s):  
Seoung Min Bong ◽  
Seung Won Yang ◽  
Ji-Woong Choi ◽  
Seung Jun Kim ◽  
Byung Il Lee
Keyword(s):  
Escherichia Coli ◽  
Polyethylene Glycol ◽  
Synchrotron Radiation ◽  
Crystallographic Analysis ◽  
Asymmetric Unit ◽  
Related Protein ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters

Myotubularin-related protein 1 is a phosphatase that dephosphorylates phospholipids such as phosphatidylinositol 3-phosphate or phosphatidylinositol 3,5-bisphosphate. In this study, human MTMR1 was overexpressed inEscherichia coli, purified and crystallized at 277 K using polyethylene glycol 20 000 as a precipitant. Diffraction data were collected to 2.0 Å resolution using synchrotron radiation. The crystals belonged to space groupP1, with unit-cell parametersa= 67.219,b= 96.587,c= 97.581 Å, α = 87.597, β = 86.072, γ = 77.327°. Assuming the presence of four molecules in the asymmetric unit, the calculated Matthews coefficient value was 2.61 Å3 Da−1and the corresponding solvent content was 52.9%.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of a single variable domain of the immunoglobulin superfamily in amphioxus,Amphi-IgSF-V

2014 ◽  
Vol 70 (8) ◽  
pp. 1072-1075 ◽  
Author(s):  
Bo Jiang ◽  
Yanjie Liu ◽  
Rong Chen ◽  
Zhenbao Wang ◽  
Mansoor Tariq ◽  
...  
Keyword(s):  
Immune System ◽  
Structural Information ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Immunoglobulin Superfamily ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
System A ◽  
Human Immunoglobulins

Amphioxus is regarded as an essential animal model for the study of immune evolution. Discovery of new molecules with the immunoglobulin superfamily (IgSF) variable (V) domain in amphioxus would help in studying the evolution of IgSF V molecules in the immune system. A protein was found which just contains only one IgSF V domain in amphioxus, termedAmphi-IgSF-V; it has over 30% sequence identity to the V domains of human immunoglobulins and mammalian T-cell receptors. In order to clarify the three-dimensional structure of this new molecule in amphioxus,Amphi-IgSF-V was expressed, purified and crystallized, and diffraction data were collected to a resolution of 1.95 Å. The crystal belonged to space groupP3221, with unit-cell parametersa=b= 53.9,c= 135.5 Å. The Matthews coefficient and solvent content were calculated to be 2.58 Å3 Da−1and 52.38%, respectively. The results will provide structural information to study the evolution of IgSF V molecules in the immune system.

scholarly journals Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6

2014 ◽  
Vol 70 (6) ◽  
pp. 794-799 ◽  
Author(s):  
Junko Morita ◽  
Kazuki Kato ◽  
Emiko Mihara ◽  
Ryuichiro Ishitani ◽  
Junichi Takagi ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Choline Metabolism ◽  
Protein Molecules ◽  
Membrane Bound

Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8 Å resolution. The crystal belonged to space groupP1, with unit-cell parametersa= 63.7,b= 68.8,c= 69.7 Å, α = 60.6, β = 87.0, γ = 68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%.

scholarly journals Preliminary crystallographic analysis of Xyn52B2, a GH52 β-D-xylosidase fromGeobacillus stearothermophilusT6

2014 ◽  
Vol 70 (12) ◽  
pp. 1675-1682 ◽  
Author(s):  
Roie Dann ◽  
Shifra Lansky ◽  
Noa Lavid ◽  
Arie Zehavi ◽  
Valery Belakhov ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Crystal Form ◽  
Thermophilic Bacterium ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Glycoside Hydrolase Family ◽  
Data Sets ◽  
X Ray Diffraction ◽  
Cell Parameters ◽  
Average Unit

Geobacillus stearothermophilusT6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of β-xylosidases. One of these β-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombicP212121space group, with average unit-cell parametersa = 97.7,b= 119.1,c = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Å resolution), the E335G catalytic mutant (2.95 Å resolution), a potential mercury derivative (2.15 Å resolution) and a selenomethionine derivative (3.90 Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

Preparation and preliminary X-ray diffraction studies of a new crystal form of L-asparaginase from Escherichia coli

1999 ◽  
Vol 55 (9) ◽  
pp. 1616-1617
Author(s):  
I. Polikarpov ◽  
R. T. de Oliveira ◽  
J. Abrahão-Neto
Keyword(s):  
Escherichia Coli ◽  
Synchrotron Radiation ◽  
Crystal Form ◽  
Asymmetric Unit ◽  
Chemotherapeutic Drug ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Synchrotron Radiation Diffraction

L-Asparaginase is an enzyme which hydrolyzes asparagine to produce aspartic acid and ammonia. It is an effective chemotherapeutic drug, especially in the treatment of acute lymphoblastic leukaemia in children. The enzyme from Escherichia coli was crystallized in a new crystal form with space group C2, unit-cell parameters a = 76.3 (0), b = 134.6 (2), c = 64.8 (7) Å, β = 110.5 (1)° and a dimer in the asymmetric unit. Synchrotron-radiation diffraction data have been collected to 1.95 Å resolution.

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