scholarly journals Purification and Crystallographic Analysis of a Novel Cold-Active Esterase (HaEst1) from Halocynthiibacter arcticus

Crystals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 170
Author(s):  
Sangeun Jeon ◽  
Jisub Hwang ◽  
Wanki Yoo ◽  
Joo Won Chang ◽  
Hackwon Do ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Primary Sequence ◽  
Unit Cell Parameters ◽  
Cell Parameters ◽  
Crystallographic Study ◽  
Primary Sequence Analysis ◽  
Sds Page ◽  
Cold Active ◽  
Zymographic Analysis ◽  
Peg 8000

This report deals with the purification, characterization, and a preliminary crystallographic study of a novel cold-active esterase (HaEst1) from Halocynthiibacter arcticus. Primary sequence analysis reveals that HaEst1 has a catalytic serine in G-x-S-x-G motif. The recombinant HaEst1 was cloned, expressed, and purified. SDS-PAGE and zymographic analysis were carried out to characterize the properties of HaEst1. A single crystal of HaEst1 was obtained in a solution containing 10% (w/v) PEG 8000/8% ethylene glycol, 0.1 M Hepes-NaOH, pH 7.5. Diffraction data were collected to 2.10 Å resolution with P21 space group. The final Rmerge and Rp.i.m values were 7.6% and 3.5% for 50–2.10 Å resolution. The unit cell parameters were a = 35.69 Å, b = 91.21 Å, c = 79.15 Å, and β = 96.9°.

scholarly journals Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the chicken CD8αα–BF2*0401 complex

2014 ◽  
Vol 70 (9) ◽  
pp. 1264-1267 ◽  
Author(s):  
Yanjie Liu ◽  
Rong Chen ◽  
Mansoor Tariq ◽  
Chun Xia
Keyword(s):  
Mhc Class I ◽  
Cytotoxic T Cells ◽  
Crystallographic Analysis ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Molecular Weights ◽  
Mhc Class I Molecule ◽  
Sds Page ◽  
Chicken Mhc

In the process of antigen presentation, the MHCI–CD8 complex is important for immune signal transduction by the activation of cytotoxic T cells. Here, the expression, purification, crystallization and X-ray analysis of the complex of the chicken MHC class I molecule BF2*0401 and CD8αα (CD8αα–BF2*0401) are reported. This complex was verified by SDS–PAGE analysis of a CD8αα–BF2*0401 crystal, which showed three bands corresponding to the molecular weights of BF2*0401,β2-microglobulin and CD8α, respectively. The crystal of CD8αα–BF2*0401 diffracted to 2.8 Å resolution and belonged to space groupP21, with unit-cell parametersa= 90.6,b= 90.8,c= 94.9 Å,β= 98°. The Matthews coefficient and solvent content were calculated to be 2.88 Å3 Da−1and ∼57.3%, respectively.

A Crystallographic Study of Bis[S-benzyl-5-bromo-2-oxoindolin-3-ylidenemethanehydrazonthioate] Disulfide Solvated with Dimethylsulfoxide

2012 ◽  
Vol 9 (2) ◽  
pp. 87
Author(s):  
Mohd Abdul Fatah Abdul Manan ◽  
M. Ibrahim M. Tahir ◽  
Karen A. Crouse ◽  
Fiona N.-F. How ◽  
David J. Watkin
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonds ◽  
Solvent Molecule ◽  
Site Occupancy ◽  
Unit Cell Parameters ◽  
Intermolecular Hydrogen Bonds ◽  
Triclinic Space Group ◽  
Cell Parameters ◽  
Crystallographic Study ◽  
Thiol Form

The crystal structure of the title compound has been determined. The compound crystallized in the triclinic space group P -1, Z = 2, V = 1839 .42( 18) A3 and unit cell parameters a= 11. 0460( 6) A, b = 13 .3180(7) A, c=13. 7321 (8) A, a = 80.659(3 )0, b = 69 .800(3 )0 and g = 77 .007 (2)0 with one disordered dimethylsulfoxide solvent molecule with the sulfur and oxygen atoms are distributed over two sites; S101/S102 [site occupancy factors: 0.6035/0.3965] and 0130/0131 [site occupancy factor 0.3965/0.6035]. The C22-S2 l and C 19-S20 bond distances of 1. 779(7) A and 1. 788(8) A indicate that both of the molecules are connected by the disulfide bond [S20-S21 2.055(2) A] in its thiol form. The crystal structure reveals that both of the 5-bromoisatin moieties are trans with respect to the [S21-S20 and CI 9-Nl 8] and [S20-S21 and C22-N23] bonds whereas the benzyl group from the dithiocarbazate are in the cis configuration with respect to [S21-S20 and C19-S44] and [S20-S21 and C22-S36] bonds. The crystal structure is further stabilized by intermolecular hydrogen bonds of N9-H35···O16 formed between the two molecules and N28-H281 ···O130, N28-H281 ···O131 and C4 l-H4 l l ···O 131 with the solvent molecule.

Synchrotron powder diffraction characterization of the zeolite-based (p-N,N-dimethylnitroaniline–mordenite) guest–host phase

2008 ◽  
Vol 64 (6) ◽  
pp. 713-724 ◽  
Author(s):  
Florence Porcher ◽  
Elena Borissenko ◽  
Mohamed Souhassou ◽  
Masaki Takata ◽  
Kenichi Kato ◽  
...  
Keyword(s):  
Powder Diffraction ◽  
Dipole Moments ◽  
Membered Ring ◽  
Unit Cell Parameters ◽  
Cell Parameters ◽  
Crystallographic Study ◽  
Synchrotron Powder Diffraction ◽  
Ring Channel ◽  
New Phase

The crystal structure of a new phase consisting of the inclusion of the hyperpolarizable molecule p-N,N-dimethylnitroaniline (dimethyl-para-nitroaniline or dmpNA) in the large-pore zeolite mordenite (MOR) has been determined from high-resolution synchrotron powder diffraction at 300 and 90 K. The unit-cell parameters and space group at 300 K are similar to those of as-synthesized mordenite. The crystallographic study indicates that the MOR straight channels are almost fully loaded with molecules that are disordered over eight symmetry-related sites. As expected, the molecules are located in the large 12-membered ring channel, at the intersection with the secondary eight-membered channel with which they might form hydrogen bonds. The elongation axes (and then the dipole moments) of the molecules are slightly tilted (28.57°) from [001]. The configuration found suggests an interaction of dmpNA with framework O atoms through its methyl groups.

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 Staphylococcus aureuslipase: purification, kinetic characterization, crystallization and crystallographic study

2018 ◽  
Vol 74 (9) ◽  
pp. 567-570 ◽  
Author(s):  
Mutsumi Tanaka ◽  
Shigeki Kamitani ◽  
Kengo Kitadokoro
Keyword(s):  
Anion Exchange Chromatography ◽  
Crystallographic Analysis ◽  
Exchange Chromatography ◽  
Structure Based Drug Design ◽  
Cell Parameters ◽  
Metal Affinity ◽  
Crystallographic Study ◽  
Diffusion Technique ◽  
Sad Phasing ◽  
Important Virulence Factor

Staphylococcus aureuslipase (SAL), a triacylglycerol esterase, is an important virulence factor inS. aureusand may be a therapeutic target for infectious diseases caused byS. aureus. For the purposes of anti-SAL drug development using structure-based drug design, X-ray crystallographic analysis of SAL overexpressed inEscherichia coliwas performed. The recombinant protein was purified using a three-step protocol involving immobilized metal-affinity chromatography, cation-exchange chromatography and anion-exchange chromatography flowthrough methods, yielding 40 mg of protein per litre of bacterial culture. Crystals were obtained using the sitting-drop vapor-diffusion technique. Diffraction data to 3.0 Å resolution were collected on the BL44XU beamline at SPring-8 at the zinc peak of 1.2842 Å for SAD phasing. The crystals belonged to space groupP4122 orP4322, with unit-cell parametersa= 131.0,b = 131.0,c = 250.6 Å, and are likely to contain four SAL molecules (408 residues) per asymmetric unit.

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 Production, crystallization and preliminary crystallographic analysis ofAllochromatium vinosumthiosulfate dehydrogenase TsdA, an unusual acidophilicc-type cytochrome

2014 ◽  
Vol 70 (10) ◽  
pp. 1424-1427 ◽  
Author(s):  
José A. Brito ◽  
André Gutierres ◽  
Kevin Denkmann ◽  
Christiane Dahl ◽  
Margarida Archer
Keyword(s):  
Crystallographic Analysis ◽  
Sulfur Cycle ◽  
Monoclinic Space Group ◽  
Asymmetric Unit ◽  
Oxidative Condensation ◽  
Unit Cell Parameters ◽  
Allochromatium Vinosum ◽  
Data Set ◽  
Purple Sulfur Bacterium ◽  
Cell Parameters

The ability to perform the very simple oxidation of two molecules of thiosulfate to tetrathionate is widespread among prokaryotes. Despite the prevalent occurrence of tetrathionate formation and its well documented significance within the sulfur cycle, little is known about the enzymes that catalyze the oxidative condensation of two thiosulfate anions. To fill this gap, the thiosulfate dehydrogenase (TsdA) enzyme from the purple sulfur bacteriumAllochromatium vinosumwas recombinantly expressed inEscherichia coli, purified and crystallized, and a crystallographic data set was collected. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa= 79.2,b= 69.9,c= 57.9 Å, β = 129.3°, contained one monomer per asymmetric unit and diffracted to a resolution of 1.98 Å.

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 Purification, crystallization and X-ray crystallographic analysis of human RAB11(S20V), a constitutively active GTP-binding form

2015 ◽  
Vol 71 (10) ◽  
pp. 1247-1250 ◽  
Author(s):  
Chang Min Kim ◽  
Jae Young Choi ◽  
Jong Hwan Yoon ◽  
Hyun Ho Park
Keyword(s):  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Gtp Hydrolysis ◽  
X Ray ◽  
Cell Parameters ◽  
Gtp Binding ◽  
His Tag ◽  
Hydrolysis Activity ◽  
Constitutively Active

RAB11, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking during endosome recycling. Substitution of Ser20 by Val20 in Rab11 [RAB11(S20V)] inhibits its GTP hydrolysis activity and produces a constitutively active GTP-binding form. In this study, the RAB11(S20V) mutant was overexpressed inEscherichia coliwith an engineered C-terminal His tag. RAB11(S20V) was then purified to homogeneity and was crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.4 Å from a crystal belonging to space groupI4, with unit-cell parametersa = 74.11,b= 74.11,c= 149.44 Å. The asymmetric unit was estimated to contain two molecules of RAB11(S20V).

scholarly journals Characterization, crystallization and preliminary X-ray crystallographic analysis of the Uba5 fragment necessary for high-efficiency activation of Ufm1

2014 ◽  
Vol 70 (6) ◽  
pp. 765-768 ◽  
Author(s):  
Shutao Xie
Keyword(s):  
High Efficiency ◽  
Crystallographic Analysis ◽  
Terminal Region ◽  
Adenylation Domain ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Multicellular Organisms

Uba5 is the smallest ubiquitin-like molecule-activating enzyme and contains an adenylation domain and a C-terminal region. This enzyme only exists in multicellular organisms. The mechanism through which the enzyme recognizes and activates ubiquitin-fold modifier 1 (Ufm1) remains unknown. In this study, Uba5 adenylation domains with different C-terminal region lengths were cloned, expressed and purified. The results of anin vitrotruncation assay suggest that Uba5 residues 57–363 comprise the minimal fragment required for the high-efficiency activation of Ufm1. Crystallization of Uba5 residues 57–363 was performed at 277 K using PEG 3350 as the precipitant, and crystals optimized by microseeding diffracted to 2.95 Å resolution, with unit-cell parametersa=b= 97.66,c= 144.83 Å, α = β = 90, γ = 120°. There is one molecule in the asymmetric unit; the Matthews coefficient and the solvent content were calculated to be 2.93 Å3 Da−1and 58.1%, respectively.

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